Compositions and methods for treating oculopharyngeal muscular dystrophy (OPMD)

Abstract

The present disclosure relates to modified adeno-associated virus (AAV) delivery vectors containing "silence and replacement" DNA constructs, compositions containing the same, and the use of the modified AAVs and compositions for treating oculopharyngeal muscular dystrophy (OPMD) in individuals suffering from or predisposed to OPMD. In particular, the present disclosure relates to AAVs having capsid proteins with modified subunit 1 (VP1) and containing "silence and replacement" DNA constructs, where the "silence and replacement" DNA constructs include (i) a DNA-directed RNAi (ddRNAi) construct encoding a short hairpin microRNA (shmiR) targeting PABPN1, a causative factor in OPMD, and (ii) a PABPN1 construct encoding a functional PABPN1 protein with an mRNA transcript not targeted by the shmiR in (i). The present disclosure also relates to a method for treating OPMD, comprising directly injecting the AAVs of the present disclosure into the pharyngeal muscles of a subject.

Description

【Technical Field】 【0001】 Cross-reference of related applications This application claims the priority of U.S. Provisional No. 62 / 812,187, filed on Feb. 28, 2019, the entire content of which is incorporated herein by reference. 【0002】 Refer to the sequence listings submitted electronically via EFS-Web. This application includes a Sequence Listing (name: "4226_0190001_SeqListing_ST25.txt"; size: 55,600 bytes; and creation date: Feb. 26, 2019) submitted electronically via EFS-Web, the entire content of which is incorporated herein by reference. 【0003】 Technical field The present disclosure relates to a modified adeno-associated virus (AAV) delivery vector comprising a "silence and substitution" DNA construct, a composition comprising the same, and the use of the modified AAV and the composition for treating oculopharyngeal muscular dystrophy (OPMD) in an individual having or predisposed to OPMD. 【Background Art】 【0004】 OPMD is an autosomal dominant hereditary, slow-progressing, late-onset degenerative muscle disorder. The disease is primarily characterized by progressive ptosis (drooping eyelids) and dysphagia (difficulty swallowing). The pharyngeal and cricopharyngeal muscles are specific targets of OPMD. Proximal limb weakness tends to follow later in the progression of the disease. The mutation causing the disease is an abnormal elongation of the (GCN)n triplet repeat in the coding region of the poly(A)-binding protein nucleus 1 (PABPN1) gene. This elongation results in an elongated polyalanine tract at the N-terminus of the PABPN1 protein. While a normal protein contains 10 alanine molecules, the mutant form (expPABPN1) has 11–18 alanine molecules. The main pathological feature of this disease is the nuclear aggregate of expPABPN1. Misfolding of the elongated PABPN1 leads to the accumulation of insoluble high-molecular-weight fibril aggregates in the nucleus of affected cells. PABPN1 is a protein prone to aggregation, and mutant alanine-extended PABPN1 within OPMD exhibits a higher aggregation rate than the wild-type normal protein. However, it remains unclear whether the nuclear aggregates within OPMD have a pathological function or a protective role as a result of cellular defense mechanisms. 【0005】 No pharmacologically or otherwise approved treatments are currently available for OPMD. Symptomatic surgical interventions can partially correct ptosis and improve swallowing in moderately to severely affected individuals. For example, cricopharyngeal myotomy is currently the only available treatment to improve swallowing in these patients. However, this does not correct the progressive deterioration of pharyngeal muscle tissue and often leads to death after dysphagia and choking. 【0006】 Therefore, therapeutic agents for treating OPMD remain necessary in patients who have OPMD and / or are predisposed to OPMD. [Overview of the project] 【0007】 This disclosure is based in part on the inventors' recognition that there are currently no approved therapeutic agents for the treatment of OPMD. Accordingly, this disclosure provides a therapeutic agent for the treatment of OPMD based on a modified adeno-associated virus (AAV) delivery vector comprising a “silence and substitution” construct, the therapeutic agent comprising (i) one or more RNAi agents targeting a region of the PABPN1 mRNA transcript that causes OPMD, and (ii) a PABPN1 substitution construct for the expression of wild-type (functional) human PABPN1 protein having an mRNA transcript that is not targeted by the RNAi agents of this disclosure. This disclosure also provides a method for treating OPMD using an AAV delivery vector and a composition comprising the same. 【0008】 For example, this disclosure provides adeno-associated viruses (AAVs) including the following: (a) AAV9-derived viral capsid protein containing a modified subunit 1 (VP1) sequence in which the amino acids at positions 1, 26, 40, 43, and 44 are modified compared to the corresponding wild-type AAV9 VP1 sequence described in Sequence ID No. 87: and (b) a polynucleotide sequence comprising a DNA-directed RNAi (ddRNAi) construct comprising a nucleic acid comprising a sequence encoding a short hairpin microRNA (shmiR); and (ii) a PABPN1 construct comprising a nucleic acid comprising a sequence encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR(s) encoded by the ddRNAi construct. 【0009】 For example, the modified AAV9 VP1 sequence contains serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and serine at position 44, compared to the AAV9 VP1 sequence described in SEQ ID NO: 87. For example, the modified AAV9 VP1 sequence may contain the following modified A1S, A26E, Q40R, K43D, and A44S, compared to the sequence described in SEQ ID NO: 87. For example, the modified AAV9 VP1 sequence contains the sequence described in SEQ ID NO: 88. 【0010】 In one example, the viral capsid protein contains mutations A42S, A67E, Q81R, K84D, and A85S with respect to the full-length wild-type AAV serotype 9 capsid sequence described in SEQ ID NO: 89. In another example, the viral capsid protein contains the amino acid sequence described in SEQ ID NO: 90. 【0011】 This disclosure also provides AAVs including: (a) AAV8-derived viral capsid protein containing a modified subunit 1 (VP1) sequence in which the amino acids at positions 1, 26, 40, 43, 44 and 64 are modified compared to the corresponding wild-type AAV8 VP1 sequence described in Sequence ID No. 91: and (b) A polynucleotide sequence comprising (i) a ddRNAi construct comprising a nucleic acid comprising a sequence encoding a shmiR; and (ii) a PABPN1 construct comprising a nucleic acid comprising a sequence encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR(s) encoded by the ddRNAi construct. 【0012】 For example, the modified AAV8 VP1 sequence contains serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, serine at position 44, and lysine at position 64, compared to the AAV8 VP1 sequence described in SEQ ID NO: 91. For example, the modified AAV8 VP1 sequence may contain the following modified A1S, A26E, Q40R, K43D, A44S, and Q64K, compared to the sequence described in SEQ ID NO: 91. For example, the modified AAV8 VP1 sequence contains the sequence described in SEQ ID NO: 92. 【0013】 In one example, the viral capsid protein contains mutations A42S, A67E, Q81R, K84D, A85S, and Q105K with respect to the full-length wild-type AAV serotype 8 capsid sequence described in SEQ ID NO: 93. In another example, the viral capsid protein contains the amino acid sequence described in SEQ ID NO: 94. 【0014】 In each of the examples described above, the modified viral capsid protein is a delivery vector for polynucleotides containing the ddRNAi construct and the PABPN1 construct. In one example, the polynucleotide sequence contains the ddRNAi construct and the PABPN1 construct in the 5' to 3' direction. In another example, the polynucleotide sequence contains the PABPN1 construct and the ddRNAi construct in the 5' to 3' direction. 【0015】 The polynucleotide may further contain reverse terminal repeats (ITRs) from the AAV serotype. For example, the ITR may be adjacent to sequences containing the ddRNAi construct and the PABPN1 construct. In some examples, the ITR originates from the AAV2 serotype (e.g., SEQ ID NO: 95 and / or SEQ ID NO: 96). 【0016】 For example, the sequence encoding the functional PABPN1 protein is codon-optimized so that its mRNA transcript is not targeted by the shmiR of the ddRNAi construct. For instance, the sequence encoding the functional PABPN1 protein could be the sequence described in Sequence ID No. 73. 【0017】 In one example, the sequences encoding the ddRNAi construct and the functional PABPN1 protein are operably ligated to a promoter located upstream of these sequences. In some examples, the promoter is a muscle-specific promoter. 【0018】 For example, each shmiR encoded by the ddRNAi construct is An effector sequence with a length of at least 17 nucleotides; Complementary arrangement of effectors; Stem-loop array; and Includes a primary microRNA (pri-miRNA) backbone; Here, the effector array is substantially complementary to the region of corresponding length in the RNA transcript described in any one of SEQ ID NOs: 1 to 13. 【0019】 In one example, at least one shmiR encoded by the ddRNAi construct is selected from the group consisting of: An shmiR comprising the effector array described in SEQ ID NO: 15 and the effector complementary array described in SEQ ID NO: 14; An shmiR comprising the effector array described in SEQ ID NO: 17 and the effector complementary array described in SEQ ID NO: 16; An shmiR comprising the effector array described in SEQ ID NO: 19 and the effector complementary array described in SEQ ID NO: 18; An shmiR comprising the effector array described in SEQ ID NO: 21 and the effector complementary array described in SEQ ID NO: 20; An shmiR comprising the effector array described in SEQ ID NO: 23 and the effector complementary array described in SEQ ID NO: 22; An shmiR comprising the effector array described in SEQ ID NO: 25 and the effector complementary array described in SEQ ID NO: 24; An shmiR comprising the effector array described in SEQ ID NO: 27 and the effector complementary array described in SEQ ID NO: 26; An shmiR comprising the effector array described in SEQ ID NO: 29 and the effector complementary array described in SEQ ID NO: 28; An shmiR comprising the effector array described in SEQ ID NO: 31 and the effector complementary array described in SEQ ID NO: 30; An shmiR comprising the effector array described in SEQ ID NO: 33 and the effector complementary array described in SEQ ID NO: 32; An shmiR comprising the effector array described in SEQ ID NO: 35 and the effector complementary array described in SEQ ID NO: 34; An shmiR comprising the effector array described in SEQ ID NO: 37 and the effector complementary array described in SEQ ID NO: 36; and An shmiR comprising the effector array described in SEQ ID NO: 39 and the effector complementary array described in SEQ ID NO: 38. 【0020】 In one particular example, a ddRNAi construct encodes a shmiR comprising the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30, wherein the shmiR comprises the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO: 38. For example, a ddRNAi construct may encode a shmiR referred to as shmiR13 as described herein and a shmiR referred to as shmiR17 as described herein. 【0021】 For example, shmiR, or each shmiR, includes the following in the 5' to 3' direction: 5' adjacent sequence of the pri-miRNA backbone; Complementary arrangement of effectors; Stem-loop array; Effector arrangement; and The 3' adjacent sequence of the pri-miRNA backbone. 【0022】 In another example, shmiR, or each shmiR, includes the following in the 5' to 3' direction: 5' adjacent sequence of the pri-miRNA backbone; Effector arrangement; Stem-loop array; Effector complementary array; and The 3' adjacent sequence of the pri-miRNA backbone. 【0023】 For example, the stem-loop sequence is the sequence described in sequence number 40. 【0024】 For example, the pri-miRNA skeleton is the pri-miR-30a skeleton. For instance, the 5' adjacent sequence of the pri-miRNA skeleton may be the sequence described in SEQ ID NO: 41, and the 3' adjacent sequence of the pri-miRNA skeleton may be the sequence described in SEQ ID NO: 42. 【0025】 In one example, a ddRNAi construct contains at least two nucleic acids, each encoding a shmiR, and each shmiR contains an effector sequence that is substantially complementary to the RNA transcript corresponding to the PABPN1 protein that causes OPMD, and each shmiR contains a different effector sequence. 【0026】 For example, each of at least two nucleic acids in a ddRNAi construct may encode a shmiR containing an effector sequence substantially complementary to a region of corresponding length in an RNA transcript described in one of sequence numbers 1, 2, 4, 7, 9, 10, and 13. For example, at least two nucleic acids in a ddRNAi construct are selected from the group consisting of: A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR2) including the effector sequence described in SEQ ID NO: 15 and the effector complement sequence described in SEQ ID NO: 14; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR3) including the effector sequence described in SEQ ID NO: 17 and the effector complement sequence described in SEQ ID NO: 16; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR5) including the effector sequence described in SEQ ID NO: 21 and the effector complement sequence described in SEQ ID NO: 20; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR9) including the effector sequence described in SEQ ID NO: 27 and the effector complement sequence described in SEQ ID NO: 26; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR13) including the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30; nucleic acids comprising or consisting of DNA sequences encoding shmiR(shmiR14) including the effector sequence described in SEQ ID NO: 33 and the effector complement sequence described in SEQ ID NO: 32; and A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR17) which includes the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO: 38. 【0027】 For example, at least two nucleic acids in a ddRNAi construct may be selected from the group consisting of: a nucleic acid containing or consisting of the DNA sequence shown in SEQ ID NO: 56 (shmiR2); a nucleic acid containing or consisting of the DNA sequence described in SEQ ID NO: 57 (shmiR3); a nucleic acid containing or consisting of the DNA sequence described in SEQ ID NO: 59 (shmiR5); a nucleic acid containing or consisting of the DNA sequence described in SEQ ID NO: 62 (shmiR9); a nucleic acid containing or consisting of the DNA sequence described in SEQ ID NO: 64 (shmiR13); a nucleic acid containing or consisting of the DNA sequence described in SEQ ID NO: 65 (shmiR14); and a nucleic acid containing or consisting of the DNA sequence described in SEQ ID NO: 68 (shmiR17). 【0028】 In one example, each of at least two nucleic acids in the ddRNAi construct encodes a shmiR containing an effector sequence substantially complementary to a region of corresponding length in the RNA transcript described in one of sequence numbers 2, 9, 10, and 13. For example, the at least two nucleic acids in the ddRNAi construct may be selected from the group consisting of: A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR3) including the effector sequence described in SEQ ID NO: 17 and the effector complement sequence described in SEQ ID NO: 16; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR13) including the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30; nucleic acids comprising or consisting of DNA sequences encoding shmiR(shmiR14) including the effector sequence described in SEQ ID NO: 33 and the effector complement sequence described in SEQ ID NO: 32; and A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR17) which includes the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO: 38. 【0029】 For example, at least two nucleic acids in a ddRNAi construct may be selected from the group consisting of: a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 57 (shmiR3); a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 64 (shmiR13); a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 65 (shmiR14); and a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 68 (shmiR17). 【0030】 In one particular example, at least two nucleic acids are selected from nucleic acids comprising or consisting of DNA sequences encoding shmiR(shmiR13) including the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30, and nucleic acids comprising or consisting of DNA sequences encoding shmiR(shmiR17) including the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO: 38. For example, at least two nucleic acids may be nucleic acids comprising or consisting of the DNA sequence described in SEQ ID NO: 64(shmiR13), and nucleic acids comprising or consisting of the DNA sequence described in SEQ ID NO: 68(shmiR17). 【0031】 In any of the examples described above, the ddRNAi construct and the PABPN1 construct can be operably ligated to a promoter. In one example, the ddRNAi construct and the PABPN1 construct are operably ligated to the same promoter, for example, a muscle-specific promoter. Compositions comprising the AAV of this disclosure and one or more pharmaceutically acceptable carriers are also provided. 【0032】 This disclosure also provides multiple baculovirus vectors for producing the AAV of this disclosure in insect cells. For example, the multiple baculovirus vectors include: (a) A first baculovirus vector comprising a nucleic acid molecule encoding an AAV virus capsid protein having the modified VP1 sequence described herein; and (b) A second baculovirus vector comprising polynucleotides encoding the ddRNAi construct and the PABPN1 construct described herein, adjacent to the AAV reverse terminal repeat (ITR) sequence. 【0033】 For example, the first baculovirus vector contains a nucleic acid molecule encoding a viral capsid protein derived from AAV9 containing a modified VP1 sequence, wherein the amino acids at positions 1, 26, 40, 43, and 44 are modified compared to the corresponding wild-type AAV9 VP1 described in Sequence ID No. 87. The second baculovirus vector contains a polynucleotide sequence comprising (i) a ddRNAi construct encoding a shmiR, and (ii) a PABPN1 construct encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR(s) encoded by the ddRNAi construct. 【0034】 For example, the modified AAV9 VP1 sequence contains serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and serine at position 44, compared to the AAV9 VP1 sequence described in SEQ ID NO: 87. For example, the modified AAV9 VP1 sequence may contain the following modified A1S, A26E, Q40R, K43D, and A44S, compared to the sequence described in SEQ ID NO: 87. For example, the modified AAV9 VP1 sequence contains the sequence described in SEQ ID NO: 88. 【0035】 In one example, the viral capsid protein contains mutations A42S, A67E, Q81R, K84D, and A85S with respect to the full-length wild-type AAV serotype 9 capsid sequence described in SEQ ID NO: 89. In another example, the viral capsid protein contains the amino acid sequence described in SEQ ID NO: 90. 【0036】 In another example, the first baculovirus vector contains a nucleic acid molecule encoding a viral capsid protein derived from AAV8 containing a modified VP1 sequence, wherein the amino acids at positions 1, 26, 40, 43, 44, and 64 are modified compared to the corresponding wild-type AAV8 VP1 sequence described in Sequence ID No. 91. The second baculovirus vector contains a polynucleotide sequence comprising (i) a ddRNAi construct encoding a shmiR, and (ii) a PABPN1 construct encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR(s) encoded by the ddRNAi construct. 【0037】 For example, the modified AAV8 VP1 sequence contains serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, serine at position 44, and lysine at position 64, compared to the AAV8 VP1 sequence described in SEQ ID NO: 91. For example, the modified AAV8 VP1 sequence may contain the following modified A1S, A26E, Q40R, K43D, A44S, and Q64K, compared to the sequence described in SEQ ID NO: 91. For example, the modified AAV8 VP1 sequence contains the sequence described in SEQ ID NO: 92. 【0038】 In one example, the viral capsid protein contains mutations A42S, A67E, Q81R, K84D, A85S, and Q105K with respect to the full-length wild-type AAV serotype 8 capsid sequence described in SEQ ID NO: 93. In another example, the viral capsid protein contains the amino acid sequence described in SEQ ID NO: 94. 【0039】 In each of the examples described above, the AAV ITR sequence may originate from the same serotype as the viral capsid protein encoded by the nucleic acid molecule in the first baculovirus vector. In another example, the AAV ITR sequence originates from a different AAV serotype, e.g., AAV2. In some examples, the ITR sequence originates from AAV serotype 2 and includes the sequence described in SEQ ID NO: 95 and / or SEQ ID NO: 96. 【0040】 As described herein, the second baculovirus vector comprises a ddRNAi construct encoding one or more shmiRs that target PABPN1. Exemplary ddRNAi constructs encoding shmiRs, such as combinations of shmiRs that target PABPN1, are described herein. For example, the second baculovirus vector may comprise a ddRNAi construct encoding shmiR13 and shmiR17, and a polynucleotide construct (e.g., the sequence described in SEQ ID NO: 73) containing a sequence encoding a functional PABPN1 protein whose mRNA transcript is codon-optimized so that it is not targeted by the shmiRs of the ddRNAi construct. For example, the second baculovirus vector may include a ddRNAi construct comprising a DNA sequence encoding a shmiR that includes the effector sequence described in SEQ ID NO: 31 and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 31, for example, the effector complement sequence described in SEQ ID NO: 30 (shmiR13), and a nucleic acid comprising a DNA sequence encoding a shmiR that includes the effector complement sequence described in SEQ ID NO: 39 and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 39, for example, the effector complement sequence described in SEQ ID NO: 38 (shmiR17). For example, the second baculovirus vector may include a ddRNAi construct comprising a DNA sequence comprising the DNA sequence described in SEQ ID NO: 64 (shmiR13), and a nucleic acid comprising the DNA sequence described in SEQ ID NO: 68 (shmiR17). 【0041】 In the example of the first baculovirus vector not encoding the AAV Rep protein, multiple baculovirus vectors may further include: (c) A third baculovirus vector comprising a polynucleotide sequence encoding at least one large AAVRep protein selected from Rep78 and Rep68 and at least one small AAVRep protein selected from Rep52 and Rep40. 【0042】 At least one of several baculovirus vectors may contain a polynucleotide encoding an assembly-activating protein (AAP). For example, a baculovirus vector encoding a capsid protein may contain a polynucleotide encoding an AAP. In another example, a baculovirus encoding a Rep protein and / or a baculovirus encoding the ddRNAi construct and the PABPN1 construct may contain a polynucleotide encoding an AAP. 【0043】 This disclosure also provides a method for producing the AAV described herein in insect cells, the method is (i) Culturing insect cells containing the multiple baculovirus vectors described herein in a culture medium under conditions sufficient to produce AAV, and optionally, (ii) recovering AAV from the culture medium and / or cells, 【0044】 In one example, this method involves co-infecting insect cells with a baculovirus vector. 【0045】 In one example, a method for producing AAV includes recovering AAV from the culture medium and / or cells. In another example, a method for producing AAV includes recovering AAV from the culture medium and / or cells, and then purifying the AAV. In one example, AAV is recovered from cells. In one example, AAV is recovered from the culture medium. In another example, AAV is recovered from both cells and the culture medium. 【0046】 This disclosure also provides AAV produced by the method described herein. 【0047】 This disclosure also provides a method for treating a subject suffering from oculopharyngeal muscular dystrophy (OPMD), the method comprising administering to the subject the AAV of this disclosure or a composition comprising the AAV of this disclosure, wherein the AAV or composition comprising the AAV of this disclosure is administered by direct injection into the subject's pharyngeal muscles. In one example, the AAV of this disclosure or a composition comprising the AAV of this disclosure is administered by direct injection into the subject's pharyngeal muscles. For example, the pharyngeal muscles include one or more of the inferior pharyngeal muscles, middle pharyngeal muscles, superior pharyngeal constrictor muscles, palatinopharynx muscles, tubal pharyngeal muscles, stylopharynx muscles, or any combination thereof. In one example, the AAV of this disclosure or a composition comprising the AAV of this disclosure is administered by direct injection into the muscles of the subject's tongue. [Brief explanation of the drawing] 【0048】 [Figure 1] A: A schematic diagram showing the construct for codon-optimized PABPN1 substitution, generated by simultaneous gene silencing of endogenous PABPN1 and subcloning two shmiRs targeting wtPABPN1 into the 3' untranslated region of a codon-optimized PABPN1 transcript between two pAAV2 ITRs (ITRs are not shown in the schematic). B: A schematic diagram showing the “silence and substitution” construct (SR construct) designed for codon-optimized PABPN1 substitution, generated by simultaneous gene silencing of endogenous PABPN1 and subcloning two shmiRs (shmiR17 and shmiR13) targeting wtPABPN1 into the 3' untranslated region of a codon-optimized PABPN1 transcript in the pAAV2 vector backbone. C: Shows the predicted secondary structure of a representative shmiR construct including the 5' facile region, siRNA sense strand; stem / loop junction sequence, siRNA antisense strand, and 3' facile region. [Figure 2]This is a schematic diagram of the SR-construction. In the SR-construction, both the "replacement" and "silence" cassettes are inserted into a single vector containing the Spc512 muscle-specific promoter. Two shmiR sequences are inserted into the 3'UTR of the codon-optimized PABPN1 cassette. [Figure 3A] This figure shows shRNA expression in the tibialis anterior muscle (TA) of A17 mice injected with SR-Construct. RNA was extracted from TA samples 14 weeks after SR-Construct administration. [Figure 3B] This figure shows the silencing of PABPN1 expression (expPABPN1, etc.) in the TA muscle of A17 mice treated with SR-Construct. RNA was extracted from TA samples 14 weeks after SR-Construct administration. [Figure 3C] This figure shows the recovery of normal PABPN1 levels in the A17 mouse model during treatment with SR-Construct. RNA was extracted from TA muscle samples 14 weeks after administration of SR-Construct. [Figure 4A] This figure shows a significant reduction in the formation of insoluble aggregates (intranuclear inclusions (INIs)) containing PABPN1, with a dose-effect relationship with SR-Construct. SR-Construct was injected into the TA muscle of A17 mice. Muscle tissue was collected and mounted for histological study 14 weeks after SR-Construct administration. Immunofluorescence of PABPN1 is shown in green, and immunofluorescence of laminin is shown in red. [Figure 4B] This shows the quantification of the proportion of nuclei containing INI in muscle sections. This indicates that treatment with SR-Construct significantly reduces the amount of INI compared to untreated A17 TA muscle (one-way ANOVA with Bonferroni post-hoc test, ***p<0.001, ns: not significant). [Figure 5A] This figure shows a significant increase in maximal force induced by the TA muscle of A17 mice using a dose-dependent method with SR-Construct. Maximal force was measured by in situ muscle physiology. [Figure 5B]This figure shows the muscle weight normalized to body weight (BW) of TA muscle treated with SR-Construct in A17 mice. The normalized muscle weight was comparable to that of control FvB mice at doses exceeding 1e10vg per injected TA (mean ± SEM n=10, one-way ANOVA with Bonferroni post-hoc test, *p<0.05, ***p<0.001, **p<0.01, ns: not significant). [Figure 6A] This figure shows the maximum force generated by the TA muscle of A17 mice 14 weeks after administration of SR-Construct. Maximum force was measured by in situ muscle physiology. [Figure 6B] This figure shows the maximum force generated by the TA muscle of A17 mice 20 weeks after administration of SR-Construct. Maximum force was measured by in situ muscle physiology. [Figure 7A] This figure shows the direct injection of SR-Construct into the pharyngeal muscle of a sheep. [Figure 7B] This figure shows radiographic images using radiolabeled cream, illustrating severe swallowing difficulties in human OPMD patients at risk of aspiration. [Figure 8] This figure shows the vector map of a DNA construct called BacAAV9-Rep-VPmod. This DNA construct is designed to express the AAVRep protein and modified AAV9 capsid in insect cells. The vector backbone is the baculovirus vector pOET1 backbone (Oxford Expression Technologies), which was used to prepare AAV containing the modified AAV9 capsid protein. [Figure 9] This figure shows the vector map of a DNA construct called AAV9-VPmod. This DNA construct contains a modified version of the AAV9 capsid gene used in the preparation of BacAAV9-Rep-VPmod (Figure 8). [Figure 10]This figure shows the vector map of a DNA construct called AAV9-Rep-VPmod. This DNA construct is designed to express the AAVRep protein and a modified AAV9 capsid in insect cells. [Figure 11] This figure shows the vector map of a DNA construct called BacAAV8-Rep-VPmod. This DNA construct is designed to express both the AAVRep protein and the modified AAV8 capsid in insect cells. The vector backbone is the baculovirus vector pOET1 backbone (Oxford Expression Technologies) and was used to prepare AAV containing the modified AAV8 capsid protein in insect cells. [Figure 12] This figure shows the vector map of a DNA construct called AAV8-VPmod. This DNA construct contains modified versions of the AAV8 capsid gene used to prepare AAV8-Rep-VPmod (Figure 13) and BacAAV8-Rep-VPmod (Figure 11). [Figure 13] This figure shows the vector map of a DNA construct called wtAAV8-Rep / Cap. This DNA construct was designed to express the AAVRep protein and wtAAV8 capsid in insect cells and was used to prepare AAV containing the wtAAV8 capsid protein. [Figure 14] This figure shows the vector map of a DNA construct called AAV2-GOI. This DNA construct was designed to express two shmiRs adjacent to the AAV ITR and was used to prepare BacAAV2-GOI (Figure 15). [Figure 15]This figure shows the vector map of a DNA construct called BacAAV2-GOI. This DNA construct was designed to express two shmiRs adjacent to the AAV ITR (AAV2-GOI) within the baculovirus vector pOET1 backbone (Oxford Expression Technologies). Using this construct, we prepared an AAV containing a modified AAV9 capsid protein expressing a GOI encoding the two shmiRs. [Figure 16] Figures A-C show the total number of expressed shmiR copies per cell from JHU67 cells infected with 4x10e9, 8x10e9, and 1.6x10e10 AAV vector genomes. (i) AAV8 (VecBio) containing unmodified VP1 produced in mammalian cells, (ii) AAV8 (BacVPmod) containing modified VP1 produced by baculovirus in insect cells, and (iii) AAV8 (Ben10) with unmodified VP1 produced by baculovirus in insect cells. AAVs with wild-type capsids produced in mammalian cells express high levels of shmiR compared to AAVs with wild-type capsids produced in insect cells, where expression is barely detectable. AAVs with capsids containing modified VP1 produced in insect cells show a significant increase in expression, and therefore functionality, compared to AAVs produced in insects using unmodified wild-type capsids. [Figure 17] This figure shows the total number of shmiR copies expressed from C2C12 cells infected with 4x10e9, 8x10e9, and 1.6x10e10 AAV vector genomes expressing the AAV endogenized receptor (AAV-R), specifically AAV9 containing unmodified VP1 produced in mammalian cells and (ii) AAV9 containing modified VP1 produced by baculovirus in insect cells. Both recombinant viruses produced comparable levels of shmiR, indicating comparable functionality. [Modes for carrying out the invention] 【0049】 Sequence Listing Symbols (KEY TO THE SEQUENCE LISTING) Sequence ID 1: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, called PABPN1 mRNA region 2. Sequence ID 2: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, known as the PABPN1 mRNA region 3. Sequence ID 3: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, known as the PABPN1 mRNA region 4. Sequence ID 4: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, known as the PABPN1 mRNA region 5. Sequence ID 5: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, known as the PABPN1 mRNA region 6. Sequence ID 6: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, known as the PABPN1 mRNA region 7. Sequence ID 7: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, known as the PABPN1 mRNA region 9. Sequence ID 8: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, called the PABPN1 mRNA region 11. Sequence ID 9: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, known as the PABPN1 mRNA region 13. Sequence ID 10: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, known as the PABPN1 mRNA region 14. Sequence ID 11: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, known as the PABPN1 mRNA region 15. Sequence ID No. 12: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, known as the PABPN1 mRNA region 16. Sequence ID 13: The RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, known as the PABPN1 mRNA region 17. Sequence ID 14: The RNA effector complement sequence of shmiR called shmiR2. Sequence ID 15: The RNA effector sequence of shmiR called shmiR2. Sequence ID 16: The RNA effector complement sequence of shmiR called shmiR3. Sequence ID 17: The RNA effector sequence of shmiR called shmiR3. Sequence ID 18: The RNA effector complement sequence of shmiR called shmiR4. Sequence ID 19: The RNA effector sequence of shmiR called shmiR4. Sequence ID 20: The RNA effector complement sequence of shmiR called shmiR5. Sequence ID 21: The RNA effector sequence of shmiR called shmiR5. Sequence ID 22: The RNA effector complement sequence of shmiR called shmiR6. Sequence ID 23: The RNA effector sequence of shmiR called shmiR6. Sequence ID 24: The RNA effector complement sequence of shmiR called shmiR7. Sequence ID 25: The RNA effector sequence of shmiR called shmiR7. Sequence ID 26: The RNA effector complement sequence of shmiR called shmiR9. Sequence ID 27: The RNA effector sequence of shmiR called shmiR9. Sequence ID 28: The RNA effector complement sequence of shmiR called shmiR11. Sequence ID 29: The RNA effector sequence of shmiR called shmiR11. Sequence ID 30: The RNA effector complement sequence of shmiR called shmiR13. Sequence ID 31: The RNA effector sequence of shmiR called shmiR13. Sequence ID 32: The RNA effector complement sequence of shmiR called shmiR14. Sequence ID 33: The RNA effector sequence of shmiR called shmiR14. Sequence ID 34: The RNA effector complement sequence of shmiR called shmiR15. Sequence ID 35: The RNA effector sequence of shmiR called shmiR15. Sequence ID 36: The RNA effector complement sequence of shmiR called shmiR16. Sequence ID 37: The RNA effector sequence of shmiR called shmiR16. Sequence ID 38: The RNA effector complement sequence of shmiR called shmiR17. Sequence ID 39: The RNA effector sequence of shmiR called shmiR17. Sequence ID 40: RNA stem-loop sequence of shmiR Sequence ID 41: 5' adjacent sequence of the pri-miRNA backbone. Sequence ID 42: 3' adjacent sequence of the pri-miRNA backbone. Sequence ID 43: The RNA sequence of shmiR called shmiR2. Sequence ID 44: The RNA sequence of shmiR called shmiR3. Sequence ID 45: The RNA sequence of shmiR called shmiR4. Sequence ID 46: The RNA sequence of shmiR called shmiR5. Sequence ID 47: The RNA sequence of shmiR called shmiR6. Sequence ID 48: The RNA sequence of shmiR called shmiR7. Sequence ID 49: The RNA sequence of shmiR called shmiR9. Sequence ID 50: The RNA sequence of shmiR called shmiR11. Sequence ID 51: The RNA sequence of shmiR called shmiR13. Sequence ID 52: The RNA sequence of shmiR called shmiR14. Sequence ID 53: The RNA sequence of shmiR called shmiR15. Sequence ID 54: The RNA sequence of shmiR called shmiR16. Sequence ID 55: The RNA sequence of shmiR called shmiR17. Sequence ID 56: A DNA sequence that encodes shmiR, called shmiR2. Sequence ID 57: A DNA sequence that encodes shmiR, called shmiR3. Sequence ID 58: A DNA sequence that codes for shmiR, called shmiR4. Sequence ID 59: A DNA sequence that encodes shmiR, called shmiR5. Sequence ID 60: A DNA sequence that encodes shmiR, called shmiR6. Sequence ID 61: A DNA sequence that encodes shmiR, called shmiR7. Sequence ID 62: A DNA sequence that encodes shmiR, called shmiR9. Sequence ID 63: A DNA sequence that encodes shmiR, called shmiR11. Sequence ID 64: A DNA sequence that codes for shmiR, called shmiR13. Sequence ID 65: A DNA sequence that codes for shmiR, called shmiR14. Sequence ID 66: A DNA sequence that encodes a shmiR called shmiR15. Sequence ID 67: A DNA sequence that encodes shmiR, called shmiR16. Sequence ID 68: A DNA sequence that encodes shmiR, called shmiR17. Sequence ID 69: A dual construct type 1 DNA sequence that encodes shmiR3 and shmiR14 under the control of the muscle-specific CK8 promoter, and codon-optimized PABPN1 under the control of Spc512. Sequence ID 70: A dual construct type 1 DNA sequence that encodes shmiR17 and shmiR13 under the control of the muscle-specific CK8 promoter, and codon-optimized PABPN1 under the control of Spc512. SEQ ID NO: 71: A double construct type 2 DNA sequence encoding coPABPN1 and shmiRs called shmiR3 and shmiR14 under the control of Spc512. Sequence ID 72: A double construct type 2 DNA sequence encoding shmiRs called coPABPN1, shmiR17, and shmiR13 under the control of Spc512. Sequence ID 73: DNA sequence of the 1-codon optimized PABPN1 cDNA sequence. Sequence ID 74: Amino acid sequence of codon-optimized human PABPN1 protein. Sequence ID 75: Amino acid sequence of wild-type human PABPN1 protein containing the FLAG tag. Sequence ID 76: Amino acid sequence of codon-optimized human PABPN1 protein containing the FLAG tag. Sequence ID 77: DNA sequence of a primer called wtPABPN1-Fwd. Sequence ID 78: DNA sequence of a primer called wtPABPN1-Rev. Sequence ID 79: DNA sequence of a probe called wtPABPN1-probe. Sequence ID 80: DNA sequence of a primer called optPABPN1-Fwd. Sequence ID 81: DNA sequence of a primer called optPABPN1-Rev. Sequence ID 82: DNA sequence of a probe called optPABPN1-probe. Sequence ID 83: DNA sequence of a primer called shmiR3-FWD. Sequence ID 84: DNA sequence of a primer called shmiR13-FWD. Sequence ID 85: DNA sequence of a primer called shmiR14-FWD. Sequence ID 86: DNA sequence of a primer called shmiR17-FWD. Sequence ID 87: A wild-type VP1 subsequence of AAV serotype 9 containing the PLA2 domain and adjacent sequences. Sequence ID 88: A modified VP1 subsequence for AAV serotype 9 containing the PLA2 domain and adjacent sequences. Sequence ID 89: Full-length wild-type AAV serotype 9 capsid. Sequence ID 90: Full-length modified AAV serotype 9 capsid. Sequence ID 91: A wild-type VP1 subsequence of AAV serotype 8 containing the PLA2 domain and adjacent sequences. Sequence ID 92: A modified VP1 subsequence of AAV serotype 8 containing the PLA2 domain and adjacent sequences. Sequence ID 93: Full-length wild-type AAV serotype 8 capsid. Sequence ID 94: Full-length modified AAV serotype 8 capsid. Sequence ID 95: AAV2 5'ITR sequence. Sequence ID 96: AAV2 3'ITR sequence. Sequence ID 97: Full-length wild-type AAV serotype 2 capsid. Sequence ID 98: RNA sequence encoding the wild-type human PABPN1 protein. 【0050】 Detailed explanation overview Throughout this Specification, unless otherwise specifically stated or the context should indicate otherwise, any reference to a single step, feature, event structure, step group, or feature or structure of an event shall be interpreted as encompassing one or more (i.e., one or more) of these steps, features, event structures, step groups, or feature or structure of an event. 【0051】 Those skilled in the art will understand that this disclosure is likely to result in variations and modifications beyond those specifically described. It should be understood that the disclosure includes all such variations and modifications. The disclosure includes all steps, features, compositions, and compounds referenced or shown individually or collectively herein, as well as all combinations or any two or more of such steps or features. 【0052】 This disclosure is not limited in scope by the specific examples described herein, which are intended to be illustrative only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure. 【0053】 Any example in this disclosure shall be taken to be applied mutatis mutandis to any other example in this disclosure, unless otherwise specified. 【0054】 Unless otherwise specifically defined, all technical and scientific terms used herein shall be interpreted in the same way as those commonly understood by those skilled in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry). 【0055】 Unless otherwise specified, the recombinant DNA, recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JEColigan et al. (editors), Current Protocols in This is described and explained in Immunology, John Wiley & Sons. 【0056】 Throughout this specification, variations of the word "comprise," "comprises," or "comprising" are understood to encompass the steps, elements, or integers, or groups of steps, elements, or integers described herein, but not to exclude any other steps, elements, or integers, or groups of steps, elements, or integers. 【0057】 The term "and / or," for example, "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as explicitly including both meanings or either of them. 【0058】 Selected definition "RNA" means a molecule containing at least one ribonucleotide residue. "Ribonucleotide" means a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranose moiety. These terms include isolated RNA such as double-stranded RNA, single-stranded RNA, and partially purified RNA, as well as essentially pure RNA, synthetic RNA, RNA produced by recombination, and modified RNA that differs from naturally occurring RNA by addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may include the addition of non-nucleotide material to the ends (or multiple ends) of an siRNA, or internally, for example, the addition of one or more nucleotides to RNA. Nucleotides in RNA molecules in this disclosure may also include non-standard nucleotides such as nucleotides that do not exist naturally or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be called analogs or analogs of naturally occurring RNA. 【0059】 The term "RNA interference" or "RNAi" generally refers to RNA-dependent silencing of gene expression initiated by double-stranded RNA (dsRNA) molecules in the cytoplasm of a cell. dsRNA molecules reduce or inhibit the transcript of a target nucleic acid sequence, thereby silencing the gene or reducing its expression. 【0060】 As used herein, the terms “double-stranded RNA” or “dsRNA” refer to an RNA molecule having a double-stranded structure and containing an effector sequence and an effector complement sequence of similar lengths. The effector sequence and effector complement sequence may be within a single RNA strand or on separate RNA strands. The “effector sequence” (often called the “guide strand”) is substantially complementary to the target sequence, which in the present invention is the region of the PABPN1 mRNA transcript. The “effector sequence” may also be referred to as the “antisense sequence.” The “effector complement sequence” is sufficiently complementary to the effector sequence so that it can anneal to the effector sequence to form a double strand. In this respect, the effector complement sequence is substantially homologous to the region of the target sequence. As will be apparent to those skilled in the art, the term “effector complement sequence” may also be called the “complement of the effector sequence” or sense sequence. 【0061】 As used herein, the term “double helix” refers to two complementary or substantially complementary regions within a nucleic acid (e.g., RNA), or two complementary or substantially complementary regions within a single-stranded nucleic acid (e.g., RNA). These are paired together by Watson-Crick base pairing, or by any other method that enables a stabilized double helix between complementary or substantially complementary nucleotide sequences. Those skilled in the art will understand that within a double helix region, 100% complementarity is not required, and substantial complementarity is acceptable. Substantial complementarity may include 79% or more complementarity. For example, a single mismatch in a double helix region consisting of 19 base pairs (i.e., 18 base pairs and one mismatch) results in 94.7% complementarity, making the double helix region substantially complementary. In another example, two mismatches in a double helix region consisting of 19 base pairs (i.e., 17 base pairs and two mismatches) result in 89.5% complementarity, making the double helix region substantially complementary. In yet another example, three mismatches in a 19-base-pair double-strand region (i.e., 16 base pairs and three mismatches) result in 84.2% complementarity, making the double-strand region substantially complementary. 【0062】 dsRNA can be provided as a hairpin or stem-loop structure having a double-stranded region containing an effector sequence and an effector complement sequence linked by at least two nucleotide sequences called a stem-loop. When dsRNA is provided as a hairpin or stem-loop structure, it can be called “hairpin RNA,” “short hairpin RNAi agent,” or “shRNA.” Other dsRNA molecules that are provided as, or cause to be provided as, a hairpin or stem-loop structure include primary miRNA transcripts (pri-miRNA) and precursor microRNAs (pre-miRNA). Pre-miRNA and shRNA can be spontaneously generated from pri-miRNA by the action of the enzymes Drosha and Pasha, which recognize and release the region of the primary miRNA transcript that forms the stem-loop structure. Alternatively, pri-miRNA transcripts can be manipulated to replace the native stem-loop structure with an artificial / recombinant stem-loop structure. That is, the artificial / recombinant stem-loop structure can be inserted into or cloned into a pri-miRNA backbone sequence that does not contain its native stem-loop structure. For stem-loop sequences designed to be expressed as part of a pri-miRNA molecule, Drosha and Pasha recognize and release the artificial shRNA. The dsRNA molecules produced using this approach are known as "shmiRNA," "shmiR," or "microRNA framework shRNA." 【0063】 As used herein, the term “complementary” with respect to sequences refers to the Watson-Crick base pair complement of a sequence, where guanine (G) is paired with cytosine (C), and adenine (A) is paired with either uracil (U) or thymine (T). A sequence may be complementary to the full length of another sequence, or to a particular portion or length of another sequence. Those skilled in the art will recognize that U may be present in RNA and T may be present in DNA. Thus, A in either an RNA or DNA sequence may be paired with U in an RNA sequence or T in a DNA sequence. Those skilled in the art will also recognize that G present in RNA may be paired with C or U in RNA. 【0064】 As used herein, the term “substantially complementary” is used to indicate a degree of complementarity or precise pairing such that stable, specific binding occurs between nucleic acid sequences, for example, between an effector sequence and an effector complement sequence, or between an effector sequence and a target sequence. It is understood that a nucleic acid sequence does not need to be 100% complementary to its target or complement sequence. The term encompasses sequences that are complementary to another sequence, except for overhangs. In some cases, a sequence is complementary to another sequence with one or two mismatches. In some cases, sequences are complementary with one mismatch. In some cases, sequences are complementary with two mismatches. In other cases, sequences are complementary with three mismatches. In yet another case, sequences are complementary with four mismatches. 【0065】 The term “encoded” as used in the context of shRNA or shmiR in this disclosure should be understood to mean shRNA or shmiR that can be transcribed from a DNA template. Therefore, the nucleic acids that encode or encode shRNA or shmiR in this disclosure will contain a DNA sequence that serves as a template for transcription of the respective shRNA or shmiR. 【0066】 The terms “DNA-directed RNAi construct” or “ddRNAi construct” refer to nucleic acids comprising a DNA sequence that, when transcribed, produces an RNAi-inducing shRNA or shmiR molecule (preferably shmiR). A ddRNAi construct may comprise nucleic acids to be transcribed as a single RNA capable of self-annealing to a stem-loop of at least two nucleotides, i.e., a hairpin structure having a double region linked by shRNA or shmiR; as a single RNA containing multiple shRNAs or shmiRs; or as multiple RNA transcripts, each capable of folding as a single shRNA or shmiR. A ddRNAi construct may be provided within a larger “DNA construct” containing one or more additional DNA sequences. For example, a ddRNAi construct may be provided within a DNA construct containing further DNA sequences encoding a functional PABPN1 protein whose mRNA transcript is codon-optimized so that it is not targeted by the shmiR of the ddRNAi construct. ddRNAi constructs and / or DNA constructs containing ddRNAi constructs may, for example, be contained within an expression vector operably linked to a promoter. 【0067】 As used herein, the terms “operatably linked” or “operatably linked” (or similar) mean that a coding nucleic acid sequence is linked to or associated with a regulatory sequence, such as a promoter, in a manner that promotes the expression of the coding sequence. Regulatory sequences include promoters, enhancers, and other expression regulatory elements recognized by the prior art and selected to direct the expression of a coding sequence. 【0068】 As used herein, the terms “reverse-end repeat” or “ITR” refer, in plural or singular, to sequences located at one end of a vector that, when used in combination with a complementary sequence located at the opposite end of the vector, can form a hairpin structure. Pairs of reverse-end repeats are involved in the rescue, replication, and packaging of AAV DNA in the host genome. ITRs are also used for the efficient capsid formation of AAV DNA and the generation of fully assembled AAV particles. 【0069】 It will be understood that “vector” means a vehicle for introducing nucleic acids into cells. Examples of vectors, but not limited to, include plasmids, phagemids, viruses, bacteria, and vehicles derived from viral or bacterial sources. A “plasmid” is a circular, double-stranded DNA molecule. A useful type of vector for use in accordance with this disclosure is a viral vector, in which a heterologous DNA sequence is inserted into a viral genome that can be modified to delete one or more viral genes or parts thereof. Some vectors can autonomously replicate within a host cell (e.g., vectors with an origin of replication that functions within a host cell). Other vectors can be stably integrated into the genome of a host cell and thereby replicate together with the host genome. As used herein, the term “expression vector” will be understood to mean a vector capable of expressing the RNA molecules of this disclosure. 【0070】 The term "functional PABPN1 protein" should be understood to mean a PABPN1 protein that possesses the functional properties of the wild-type PABPN1 protein, such as the ability to control the site of mRNA polyadenylation and / or intron splicing in mammalian cells. Therefore, a "functional PABPN1 protein" would be understood to be a PABPN1 protein that, when expressed or present in a subject, does not cause OPMD. For example, references to "functional PABPN1 protein" in this specification refer to the human wild-type PABPN1 protein. The sequence of the human wild-type PABPN1 protein is listed in NCBI RefSeq NP_004634. Therefore, a functional human PABPN1 protein may possess the in vivo functional properties of the human PABPN1 protein listed in NCBI RefSeq NP_004634. 【0071】 As used herein, the terms “treating,” “treat,” or “treatment” refer to a clinical intervention designed to alter the natural course of an individual or cell being treated during the course of clinical pathology. Desired effects of treatment include slowing the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. Therefore, treatment of OPMD includes reducing or inhibiting the expression of the PABPN1 protein that causes OPMD in the subject, and / or expressing a PABPN1 protein with polyalanine residues of normal length in the subject. Preferably, treatment of OPMD includes reducing or inhibiting the expression of the PABPN1 protein that causes OPMD in the subject, and expressing a PABPN1 protein with polyalanine residues of normal length in the subject. For example, if one or more of the above treatment outcomes are achieved, the individual is successfully “treated.” 【0072】 The “therapeutic effective dose” is the minimum concentration or amount required to produce a measurable improvement in the OPMD condition, such as, for example, a measurable improvement in one or more symptoms of OPMD, including ptosis, dysphagia, and muscle weakness, in the subject. The therapeutic effective dose specified herein may vary depending on factors such as the patient’s condition, age, sex, and weight, as well as the ability of shmiR, the nucleic acid encoding shmiR, the ddRNAi construct, the DNA construct, the expression vector, or a composition containing these, to induce a desired response in the individual and / or the ability of the expression vector to express a functional PABPN1 protein in the subject. The therapeutically effective dose is also such that the therapeutically beneficial effects of shmiR, the nucleic acid encoding it, the ddRNAi construct, the DNA construct, the expression vector, or any composition containing them outweigh any toxic or adverse effects of shmiR, the nucleic acid encoding it, the ddRNAi construct, the DNA construct, the expression vector, or any composition containing them, thereby inhibiting, suppressing, or reducing the expression of the PABPN1 protein that causes OPMD, either alone or in combination with the therapeutically beneficial effects of functional PABPN1 protein expression in the subject. 【0073】 As used herein, “subject” or “patient” may be a human or non-human animal that has OPMD or is genetically predisposed to it, i.e., has the PABPN1 gene variant that causes OPMD. “Non-human animal” may be a primate, livestock (e.g., sheep, horses, cattle, pigs, donkeys), companion animals (e.g., pets such as dogs and cats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, nematodes, zebrafish), performance animals (e.g., racehorses, camels, greyhounds), or wild animals in captivity. In one example, the subject or patient is a mammal. In one example, the subject or patient is a human. 【0074】 "Reduced expression," "reduction in expression," or similar terms refer to the absence or observable reduction in the level of mRNA product from a protein and / or target gene, e.g., the PABPN1 gene. The reduction does not need to be absolute, but may be partial enough to produce a detectable or observable change as a result of RNAi affected by shmiR, the nucleic acid encoding shmiR, a ddRNAi construct, a DNA construct, an expression vector, or a composition containing them as disclosed herein. The reduction can be measured by determining the decrease in the level of protein product from the target nucleic acid compared to cells without mRNA, and / or shmiR, the nucleic acid encoding shmiR, a ddRNAi construct, a DNA construct, an expression vector, or a composition containing them, and may be as small as 1%, 5%, or 10%, or absolute, i.e., 100% inhibition. The effect of the reduction may be determined by examining outward characteristics, i.e., the quantitative and / or qualitative phenotype of the cell or organism, and may also include detecting the presence or change in the amount of expPABPN1 nuclear aggregates in the cell or organism after administration of the shmiR, the nucleic acid encoding shmiR, the ddRNAi construct, the DNA construct, the expression vector, or the composition comprising them, as disclosed herein. 【0075】 As used herein, “delivery system” refers to a vector that can be introduced into a cell for packaging foreign genetic material, such as DNA or RNA. Delivery systems may include viral vectors, such as adeno-associated virus (AAV) vectors, retroviral vectors, adenovirus (AdV) vectors, and lentivirus (LV) vectors. As described herein, viral vectors can be used to deliver and express foreign genetic material within cells. Therefore, viral expression vectors described herein may be used as delivery systems. 【0076】 As used herein, the terms “adeno-associated virus” or “AAV” refer to a group of viruses within the Parvoviridae family that contain a short (approximately 4.7 kb) single-stranded DNA genome and depend on the presence of a helper virus, such as an adenovirus, for their replication. Furthermore, the present disclosure intends to provide, for example, AAV-derived vectors used as gene transfer vehicles. 【0077】 As used herein, the term “serotype” in the context of AAV is a distinction used to refer to an AAV having a serologically different capsid from other AAV serotypes. Serological characteristics are determined based on the absence of cross-reactivity between antibodies against one AAV compared to another AAV. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). 【0078】 When used herein in the context of AAV, “viral capsid protein,” “capsid protein,” “capsid polypeptide,” or similar terms refer to the polypeptide of AAV that has self-assembling activity to produce the proteinaceous shell of the AAV particle, also called the coat protein or VP protein. This consists of three subunits, VP1, VP2, and VP3, which are typically expressed from a single nucleic acid molecule and interact together to form an icosahedral-symmetric capsid. The capsid structure of AAV is described in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). 【0079】 As used herein, the term “promoter” generally refers to a DNA sequence that is involved in the recognition and binding of DNA-dependent RNA polymerase and other proteins (trans-acting transcription factors), initiates and controls the transcription of one or more coding sequences, and is generally located upstream of the coding sequence with respect to the direction of transcription. 【0080】 As used in the context of the AAVs of this disclosure comprising a modified capsid protein or VP1 sequence, the term “improved functionality” or similar terms should be understood to mean that the AAV comprising the modified capsid protein or VP1 sequence has improved endosomal escape activity compared to unmodified, wild-type AAV of the same serotype produced in insect cells. As used herein, “endosomal escape activity,” “endosome escape activity,” or similar terms should be understood to mean the ability of an AAV to escape from an endosomal compartment after intracellularization. In the context of AAV functionality, an AAV that cannot escape from an endosome after intracellularization will be understood to be non-functional, particularly in the context of gene therapy. 【0081】 As used herein, “pharyngeal muscles” refers to one or more of the group of muscles that form the pharynx. The pharyngeal muscles may include one or more of the inferior pharyngeal muscles, oropharynx muscles, superior pharyngeal muscles, palatinopharynx muscles, tubopharynx muscles, and / or stylopharynx muscles. 【0082】 Modified AAV delivery vector for the treatment of OPMD Adeno-associated viruses (AAVs) are dependent parvoviruses, which generally require co-infection with another virus (typically an adenovirus or herpesvirus) to initiate and maintain a productive infection cycle. In the absence of such a helper virus, AAVs still have the ability to infect or transmit to target cells via receptor-mediated binding and internalization, and to enter the nucleus within both non-dividing and dividing cells. In the absence of a helper virus, progeny viruses are not produced from AAV infection, so the scope of transduction is limited to only the initial cells infected with the virus. This characteristic makes AAVs a desirable vector for use in gene therapy. Furthermore, unlike retroviruses, adenoviruses, and herpes simplex viruses, AAVs are considered non-pathogenic and non-toxic to humans (Kay, et al., Nature. 424:251 (2003)). Not surprising, given that their genomes typically encode only two genes, AAVs are limited as a delivery medium by their packaging capacity of 4.5 kilobases (kb). However, this size limitation may restrict the genes that can be delivered for substitution gene therapy, but it does not adversely affect the packaging and expression of short sequences such as shmiR and shRNA. For these reasons, this disclosure intends to use AAV as a vector or system for delivering the PABPN1 “silence and substitution” construct for the treatment of OPMD. Generally, AAV used in gene therapy applications is preferably selected from AAV (or its variants) selected from the group consisting of human-infectious serotypes, e.g., AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. 【0083】 For example, this disclosure provides an AAV that includes: (a) A viral capsid protein containing a modified VP1 sequence in which specific amino acids and the adjacent sequences of subunit 1 (VP1) within the phospholipase A2 (PLA2) domain are modified to be more "AAV2-like" than the corresponding wild-type sequence; and (b) A polynucleotide sequence comprising (i) a ddRNAi construct comprising nucleic acid comprising a sequence encoding a short hairpin microRNA (shmiR); and (ii) a PABPN1 construct comprising nucleic acid comprising a sequence encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR(s) encoded by the ddRNAi construct. 【0084】 In this regard, the inventors have shown that the endosomal escape activity of representative AAVs from serotypes other than serotype 2 produced from baculovirus expression systems in insect cells can be restored or improved by amino acid substitutions at specific sites within the PLA2 domain and its adjacent sequences. For example, the inventors have shown that the endosomal escape activity of AAVs from representative serotypes other than serotype 2 can be restored or improved by substituting amino acids at up to six different positions within the PLA2 domain and adjacent sequences with amino acids at corresponding positions within the AAV serotype 2 PLA2 domain and adjacent sequences. In this regard, the inventors have shown that it is not necessary to replace the entire PLA2 domain and adjacent sequences with those of AAV2 to generate chimeric AAVs, nor is it necessary to generate AAVs expressing a mosaic capsid containing a wild-type VP1 / PLA2 sequence. Furthermore, unlike strategies previously employed to improve the function of AAVs produced in insect cells, it is not necessary to produce AAVs that express mosaic capsids containing wild-type VP1 / PLA2 sequences and AAV2 sequences (e.g., AAV2 / WT VP1). 【0085】 The AAV sequences that can be used to produce AAVs having the modified VP1 sequence described herein may be derived from the genome of any AAV serotype. Generally, AAV serotypes have genomic sequences that are significantly homologous at the amino acid and nucleic acid levels, provide the same set of genetic functions, produce physically and functionally similar virions, and replicate and assemble by substantially the same mechanisms (with the specific exception of the activity of the PLA2 domain described herein). Nucleic acid and protein sequences suitable for AAVs to be used in the design and production of the modified AAVs of this disclosure are publicly available. The VP1 sequence of wild-type AAV known to infect humans (and intended herein) is described in Chen et al., (2013) J.Vir. 87(11):6391-6405. Human or simian adeno-associated virus (AAV) serotypes are preferred sources of AAV nucleotide sequences for use in the context of this disclosure, more preferably AAV serotypes that normally infect humans (e.g., serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13). The capsid polypeptide sequences of AAV serotypes 1-13 are publicly known in the art, for example, AAV1 (Genbank Acc.No:AAD27757.1,GI:4689097), AAV2 (Genbank Acc.No:AAC03780.1,GP.2906023), AAV3 (Genbank Acc.No:AAC55049.1,GI:1408469), AAV4 (Genbank Acc.No:AAC58045.1,GL2337940), AAV5 (Genbank Acc.No:AAD13756.1,GI-4249658), AAV10 (Genbank Acc.No:AAT46337.1,GL48728343), AAV11 (Genbank AAV12 (Genbank Acc.No: AAT46339.1, GI: 48728346), AAV12 (Genbank Acc.No: ABI16639.1, GI: 112379656), or AAV13 (Genbank Acc.No: ABZ10812.1, GI: 167047087). The polypeptide sequences of AAV capsid proteins of serotypes 1-13 are also described in SEQ ID NOs. 27-39 herein.Furthermore, complete genomes of AAVs derived from serotypes 1-13 are publicly known in the art, for example, AAV1 (NCBI reference sequence: NC_002077.1), AAV2 (GenBank Acc. No: J01901.1), AAV3 (Genbank Acc. No: AF028705.1), AAV4 (NCBI reference sequence: NC_001829.1), AAV5 (NCBI reference sequence: NC_006152.1), AAV6 (GenBank: AF028704.1), AAV7 (NCBI reference sequence: NC_006260.1), AAV8 (NCBI reference sequence: NC_006261.1), AAV9 (GenBank Acc. No: AY530579.1), AAV10 (Genbank Acc. No: AY631965.1), AAV11 (Genbank Acc.No:AY631966.1), AAV12 (Genbank Acc.No:DQ813647.1). In certain cases, this disclosure provides AAV delivery vectors derived from serotypes 8 and 9. 【0086】 For example, the AAV of this disclosure comprises a viral capsid protein derived from AAV9 containing a modified VP1 sequence, where one or more amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence described in Sequence ID No. 87. For example, the AAV of this disclosure may comprise a viral capsid protein derived from AAV9 containing a modified VP1 sequence containing one or more of the following: serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, serine at position 44, and / or lysine at position 64, where the amino acid positions are defined relative to the wild-type AAV9 VP1 sequence shown in Sequence ID No. 87, where any one or more amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence. In some examples, any one or more additional amino acids other than those at positions 1, 26, 40, 43, and 44 are not modified compared to the corresponding wild-type AAV9 VP1 sequence. 【0087】 For example, the AAV described herein may include a viral capsid protein derived from AAV9 having a modified VP1 sequence, where any two, three, four, or five amino acids at positions 1, 26, 40, 43, and 44 are modified from the corresponding wild-type AAV9 VP1 sequence described in Sequence ID No. 87. 【0088】 For example, the AAV described herein may include a viral capsid protein derived from AAV9 having a modified VP1 sequence, where any two or more amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence described in Sequence ID No. 87. For example, the modified VP1 sequence may include two or more modifications selected from A1S, A26E, Q40R, K43D, and A44S relative to the sequence described in Sequence ID No. 87. 【0089】 For example, the AAV described herein may include a viral capsid protein derived from AAV9 having a modified VP1 sequence, where any three or more amino acids at positions 1, 26, 40, 43, and 44 are modified from the corresponding wild-type AAV9 VP1 sequence described in SEQ ID NO: 87. For example, the modified VP1 sequence may include three or more modifications selected from A1S, A26E, Q40R, K43D, and A44S with respect to the sequence described in SEQ ID NO: 87. 【0090】 For example, the AAV described herein may include a viral capsid protein derived from AAV9 having a modified VP1 sequence, where any four or more amino acids at positions 1, 26, 40, 43, and 44 are modified from the corresponding wild-type AAV9 VP1 sequence described in SEQ ID NO: 87. For example, the modified VP1 sequence may include four or more modifications selected from A1S, A26E, Q40R, K43D, and A44S with respect to the sequence described in SEQ ID NO: 87. 【0091】 For example, the AAV described herein may include a viral capsid protein derived from AAV9 having a modified VP1 sequence, where the amino acids at positions 1, 26, 40, 43, and 44 are modified compared to the corresponding wild-type AAV9 VP1 sequence described in SEQ ID NO: 87. For example, the modified VP1 sequence may include the following modified A1S, A26E, Q40R, K43D, and A44S compared to the sequence described in SEQ ID NO: 87. For example, the modified AAV9 VP1 sequence may include the amino acid sequence described in SEQ ID NO: 88. For example, the residues at positions 42, 67, 81, 84, and 85 are modified compared to the corresponding full-length wild-type AAV9 capsid VP1 sequence described in SEQ ID NO: 89 (for example, modified A42S, A67E, Q81R, K84D, and A85S compared to the sequence described in SEQ ID NO: 89). According to this embodiment, the AAV of this disclosure may include a viral capsid protein derived from AAV9, which contains the modified VP1 sequence described in SEQ ID NO: 90. 【0092】 For example, the AAV of this disclosure comprises a viral capsid protein derived from AAV8 containing a modified VP1 sequence, where one or more amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence described in SEQ ID NO: 91. For example, the AAV of this disclosure may comprise a viral capsid protein derived from AAV8 containing a modified VP1 sequence containing any one or more of serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, serine at position 44, and / or lysine at position 64, where the amino acid positions are defined relative to the wild-type AAV8 VP1 sequence described in SEQ ID NO: 91, where one or more amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence. In some examples, any one or more additional amino acids other than those at positions 1, 26, 40, 43, 44, and 64 are not modified compared to the corresponding wild-type AAV8 VP1 sequence. 【0093】 For example, the AAV described herein may include a viral capsid protein derived from AAV8 having a modified VP1 sequence, where any two, three, four, or five amino acids at positions 1, 26, 40, 43, 44, and 64 are modified from the corresponding wild-type AAV8 VP1 sequence described in Sequence ID No. 91. 【0094】 For example, the AAV described herein may include a viral capsid protein derived from AAV8 having a modified VP1 sequence, where any two or more amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence described in SEQ ID NO: 91. For example, the modified VP1 sequence may include two or more modifications selected from A1S, A26E, Q40R, K43D, A44S, and Q64K relative to the sequence described in SEQ ID NO: 91. 【0095】 For example, the AAV described herein may include a viral capsid protein derived from AAV8 having a modified VP1 sequence, where any three or more amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence described in SEQ ID NO: 91. For example, the modified VP1 sequence may include three or more modifications selected from A1S, A26E, Q40R, K43D, A44S, and Q64K relative to the sequence described in SEQ ID NO: 91. 【0096】 For example, the AAV described herein may include a viral capsid protein derived from AAV8 having a modified VP1 sequence, where any four or more amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence described in SEQ ID NO: 91. For example, the modified VP1 sequence may include four or more modifications selected from A1S, A26E, Q40R, K43D, A44S, and Q64K relative to the sequence described in SEQ ID NO: 91. 【0097】 For example, the AAV described herein may include a viral capsid protein derived from AAV8 having a modified VP1 sequence, where any five or more amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence described in SEQ ID NO: 91. For example, the modified VP1 sequence may include five or more modifications selected from A1S, A26E, Q40R, K43D, A44S, and Q64K relative to the sequence described in SEQ ID NO: 91. 【0098】 For example, the AAV described herein may include a viral capsid protein derived from AAV8 having a modified VP1 sequence, where the amino acids at positions 1, 26, 40, 43, 44, and 64 are modified compared to the corresponding wild-type AAV8 VP1 sequence described in SEQ ID NO: 91. For example, the modified VP1 sequence may include the following modified A1S, A26E, Q40R, K43D, A44S, and Q64K compared to the sequence described in SEQ ID NO: 91. For example, the modified AAV8 VP1 sequence may include the amino acid sequence described in SEQ ID NO: 92. For example, the residues at positions 42, 67, 81, 84, 85, and 105 are modified compared to the corresponding full-length wild-type AAV8 capsid VP1 sequence described in SEQ ID NO: 93 (for example, modified A42S, A67E, Q81R, K84D, A85S, and Q105K compared to the sequence described in SEQ ID NO: 93). According to this embodiment, the AAV of this disclosure may include a viral capsid protein derived from AAV8, which contains the modified VP1 sequence described in SEQ ID NO: 94. 【0099】 In each of the examples described above, the viral capsid protein may contain subunit 2 (VP2) and subunit 3 (VP3) sequences derived from the same AAV serotype as modified VP1. Preferably, VP1, VP1, and VP3 are expressed from the same ORF. 【0100】 The AAV genome includes replication (Rep) genes, which are virally encoded proteins that function in the replication of the viral genome. Thus, in one example, the AAV described herein comprises at least one large AAV Rep protein selected from Rep78 and Rep68, and at least one small AAV Rep protein selected from Rep52 and Rep40. In one example, the AAV described herein comprises Rep78 and Rep52. In one example, the AAV described herein comprises Rep78 and Rep40. In one example, the AAV described herein comprises Rep68 and Rep52. In one example, the AAV described herein comprises Rep68 and Rep40. In one example, the AAV described herein comprises Rep78, Rep68, Rep52, and Rep40. In each of the above examples, each small Rep protein and large Rep protein may originate from the same AAV serotype as the viral capsid protein. Alternatively, each small Rep protein and large Rep protein can originate from an AAV serotype other than the serotype of the viral capsid protein; for example, the Rep protein can originate from AAV2. 【0101】 As described herein, AAV can be used as a delivery system in gene therapy. For example, AAV may include a polynucleotide encoding a protein or RNA of interest. As described herein, the AAV of this disclosure includes a polynucleotide sequence comprising a ddRNAi construct and a PABPN1 construct. The polynucleotides encoding the ddRNAi construct and the PABPN1 construct may be flanked by an AAV reverse-terminus repeat (ITR) sequence. In one example, the AAV ITR sequence is derived from the same serotype as the viral capsid protein. In another example, the AAV ITR sequence is derived from a serotype other than the serotype of the viral capsid protein. In one particular example, the ITR sequence is derived from AAV serotype 2. In another particular example, the ITR sequence is derived from AAV serotype 2 and includes the sequence described in SEQ ID NO: 91 and / or SEQ ID NO: 92. 【0102】 As described above, polynucleotides encoding the target protein or RNA, including adjacent ITRs, are typically 5,000 nucleotides (nt) or less in length. However, polynucleotides encoding extra-large DNA, i.e., DNA longer than 5,000 nt, are also intended. Extra-large DNA is understood herein as DNA exceeding the 5 kbp maximum AAV packaging limit. Thus, the AAVs of this disclosure can and may be capable of expressing proteins or RNA encoded by genomes typically larger than 5.0 kb. 【0103】 As described herein, the AAVs of this disclosure also include polynucleotide sequences comprising ddRNAi constructs and PABPN1 constructs for expression in mammalian cells, which are incorporated into their genomes. Exemplary ddRNAi constructs and PABPN1 constructs are described herein (for example, under the subheading "ddRNAi constructs") and, unless otherwise specified, are to be used mutatis mutandis for the examples described relating to the AAVs of this disclosure. In this regard, the AAVs of this disclosure may include polynucleotides comprising ddRNAi constructs encoding any one or more shmiRs referred to herein as shmiR2-shmiR7, shmiR9, shmiR11, or shmiR13-shmiR17. However, in certain examples, the AAV of this disclosure may include a polynucleotide comprising a ddRNAi construct encoding shmiR13 and / or shmiR17, and a polynucleotide construct (e.g., the sequence described in SEQ ID NO: 73) comprising a sequence encoding a functional PABPN1 protein whose mRNA transcript is codon-optimized so that it is not targeted by the shmiR of the ddRNAi construct. Exemplary ddRNAi constructs encoding shmiR13 and shmiR17 are described and intended herein. 【0104】 In one particular example, AAV includes: (a) a viral capsid protein derived from AAV9 (e.g., a modified VP1 sequence containing the sequence described in SEQ ID NO: 88) having modified A1S, A26E, Q40R, K43D, and A44S compared to the corresponding wild-type sequence described in SEQ ID NO: 87; (b) a polynucleotide sequence including (i) a ddRNAi construct containing nucleic acid containing sequences encoding shmiR13 and shmiR17 as described herein, and (ii) a PABPN1 construct containing nucleic acid containing sequences encoding a functional PABPN1 protein having mRNA transcripts not targeted by the shmiR(plural) encoded by the ddRNAi construct (e.g., a codon-optimized sequence described in SEQ ID NO: 73). The polynucleotide in (b) may be adjacent to AAV reverse terminal repeat (ITR) sequences from AAV2 as described in SEQ ID NO: 95 and SEQ ID NO: 96. In some embodiments, the ddRNAi construct includes a nucleic acid comprising a DNA sequence encoding a shmiR that is substantially complementary to the effector sequence described in SEQ ID NO: 31, for example, the effector complement sequence described in SEQ ID NO: 30 (shmiR13), and a nucleic acid comprising a DNA sequence encoding a shmiR that is substantially complementary to the effector sequence described in SEQ ID NO: 39, for example, the effector complement sequence described in SEQ ID NO: 38 (shmiR17). For example, the ddRNAi construct according to this example may include a nucleic acid comprising a DNA sequence described in SEQ ID NO: 64 (shmiR13), and a nucleic acid comprising a DNA sequence described in SEQ ID NO: 68 (shmiR17). 【0105】 In another specific example, AAV includes: (a) a viral capsid protein derived from AAV8 (e.g., a modified VP1 sequence containing the sequence described in SEQ ID NO: 92) having modified A1S, A26E, Q40R, K43D, A44S, and Q64K compared to the corresponding wild-type sequence described in SEQ ID NO: 91; (b) a polynucleotide sequence including (i) a ddRNAi construct containing nucleic acids containing sequences encoding shmiR13 and shmiR17 as described herein, and (ii) a PABPN1 construct containing nucleic acids containing sequences encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR(plural) encoded by the ddRNAi construct (e.g., a codon-optimized sequence described in SEQ ID NO: 73). The polynucleotide in (b) may be adjacent to AAV reverse terminal repeat (ITR) sequences from AAV2 as described in SEQ ID NO: 95 and SEQ ID NO: 96. The polynucleotide in (b) may be adjacent to the AAV reverse terminal repeat (ITR) sequence from AAV2 as described in SEQ ID NOs. 95 and SEQ ID NOs. 96. In some embodiments, the ddRNAi construct includes or comprises a nucleic acid comprising a DNA sequence encoding a shmiR, including the effector sequence described in SEQ ID NOs. 31 and an effector complement sequence substantially complementary to the sequence described in SEQ ID NOs. 31, e.g., the effector complement sequence described in SEQ ID NOs. 30 (shmiR13); and a nucleic acid comprising or comprising an effector complement sequence substantially complementary to the sequence described in SEQ ID NOs. 39, e.g., an effector complement sequence substantially complementary to the sequence of SEQ ID NOs. 38 (shmiR17). For example, the ddRNAi construct according to this example may include or comprise a nucleic acid comprising the DNA sequence described in SEQ ID NOs. 64 (shmiR13); and a nucleic acid comprising or comprise a DNA sequence described in SEQ ID NOs. 68 (shmiR17). 【0106】 In each of the examples described above, the polynucleotides encoding the ddRNAi construct and the PABPN1 construct are operably ligated to one or more promoters suitable for the expression of the shmiR and PABPN1 proteins in mammalian cells. In one example, the promoter may be a muscle-specific promoter. Suitable muscle-specific promoters are described herein. 【0107】 The AAVs of this disclosure may comprise one large AAV Rep protein selected from Rep78 and Rep68, and at least one small AAV Rep protein selected from Rep52 and Rep40. 【0108】 In this regard, the AAV genome contains Rep genes (i.e., Rep78 and Rep52), and the proteins encoded by these genes function in the replication of the viral genome. Splicing events at the Rep ORF result in the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, unspliced ​​mRNA encoding the Rep78 and Rep52 proteins in insect cells has been shown to be sufficient for the production of AAV vectors. Therefore, in one example, AAV contains at least one large AAV Rep protein selected from Rep78 and Rep68, and at least one small AAV Rep protein selected from Rep52 and Rep40. In one example, AAV contains Rep78 and Rep52. In one example, AAV contains Rep78 and Rep40. In one example, AAV contains Rep68 and Rep52. In one example, AAV contains Rep68 and Rep40. For example, AAV includes Rep78, Rep68, Rep52, and Rep40. In each of the above examples, the smaller and larger Rep proteins may originate from the same AAV serotype as the viral capsid protein. Alternatively, the smaller and larger Rep proteins may originate from an AAV serotype other than the serotype of the viral capsid protein; for example, the Rep protein may originate from AAV serotype 2. In this regard, Rep sequences are particularly conserved among most serotypes, and it has been reported that Rep sequences efficiently cross-complement each other in insect cells. 【0109】 Any nucleotide sequence can be incorporated for subsequent expression in mammalian cells transfected with the AAV of this disclosure, as long as the construct remains within the packaging capacity of the AAV virion. 【0110】 As described herein, the AAVs described herein may be functionally improved compared to AAVs containing the corresponding wild-type VP1 sequence when produced in insect cells. 【0111】 Method and reagents for preparing AAV with modified VP1 Methods for producing AAV are known in the art. As described, the AAV of this disclosure has improved functionality (e.g., improved endosomal escape activity) when produced in insect cells compared with AAV containing the corresponding wild-type VP1 sequence. Therefore, methods and reagents for producing AAV in insect cells are intended. In some cases, insect cell-compatible vectors, i.e., baculovirus vectors, may be used to produce the AAV of this disclosure. 【0112】 For example, the present disclosure provides multiple baculovirus vectors for producing the AAV of the present disclosure in insect cells. The multiple baculovirus vectors may include: (i) A first baculovirus vector comprising a nucleic acid molecule encoding an AAV virus capsid protein having the modified VP1 sequence described herein; and (ii) A second baculovirus vector comprising polynucleotides encoding the ddRNAi construct and the PABPN1 construct described herein, adjacent to the AAV reverse terminal repeat (ITR) sequence. 【0113】 In one example, the AAV ITR sequence may originate from the same serotype as the viral capsid protein encoded by the nucleic acid molecule in the first baculovirus vector. In another example, the AAV ITR sequence originates from a different AAV serotype, e.g., AAV2. In some examples, the ITR sequence originates from AAV serotype 2 and includes the sequence described in SEQ ID NO: 95 and / or SEQ ID NO: 96. 【0114】 In some examples, the AAV comprises a capsid protein from AAV9 containing a modified VP1, as described herein. In other examples, the AAV comprises a capsid protein from AAV8 containing a modified VP1, as described herein. Thus, the first baculovirus vector may comprise a nucleic acid molecule encoding a viral capsid protein from AAV8 or AAV9 having a modified VP1 sequence. The modified VP1 sequences of the AAVs comprising the capsid proteins from AAV9 or AAV8 are described herein and, unless otherwise specified, are intended to be used mutatis mutandis in the examples of the disclosure describing baculovirus vectors for producing the AAVs of this disclosure. 【0115】 As described herein, the second baculovirus vector comprises a ddRNAi construct encoding one or more shmiRs that target PABPN1. Exemplary ddRNAi constructs encoding shmiRs, such as combinations of shmiRs that target PABPN1, are described herein and, unless otherwise specified, are intended to be used mutatis mutandis in the examples of the present disclosure describing baculovirus vectors for producing the AAV of the present disclosure. In one particular example, the second baculovirus vector may comprise a ddRNAi construct encoding shmiR13 and shmiR17, and a polynucleotide construct (e.g., the sequence described in SEQ ID NO: 73) containing a sequence encoding a functional PABPN1 protein whose mRNA transcript is codon-optimized so that it is not targeted by the shmiRs of the ddRNAi construct. For example, the second baculovirus vector may include a ddRNAi construct comprising or consisting of the effector sequence described in SEQ ID NO: 31 and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 31, for example, a DNA sequence encoding a shmiR including the effector complement sequence described in SEQ ID NO: 30 (shmiR13), and a nucleic acid comprising or consisting of the effector sequence described in SEQ ID NO: 39 and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 39, for example, a DNA sequence encoding a shmiR including the effector complement sequence described in SEQ ID NO: 38 (shmiR17). For example, the second baculovirus vector may include a ddRNAi construct comprising or consisting of the DNA sequence described in SEQ ID NO: 64 (shmiR13), a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 64, and a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 68 (shmiR17). 【0116】 In each of the aforementioned examples, the polynucleotides encoding the ddRNAi construct and the PABPN1 construct can be operably ligated to a promoter. In one example, the promoter may be a muscle-specific promoter. 【0117】Similarly, nucleic acid molecules encoding AAV virus capsid proteins can be operably ligated to promoters suitable for the expression of capsid proteins in insect cells. Promoters suitable for expression in insect cells are known in the art and are intended for use herein.In this regard, molecular engineering and methodologies for polypeptide expression in insect cells have been previously discussed, for example, in Summers and Smith, A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull No. 7555, College Station, Tex. (1986); Luckow, In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152 (1991); King, LA and RDPossee, The baculovirus expression system, Chapman and Hall, United Kingdom (1992); O'Reilly, DR, LK Miller, VA, Luckow, Baculovirus Expression Vectors: A Laboratory Manual, New York (1992); WH Freeman and Richardson, CD, Baculovirus Expression Protocols, Methods in Molecular Biology, volume It is described in 39(1992); U.S. Patent No. 4,745,051; US2003148506; WO2003 / 074714; Kotin RM(2011)Hum.Mol.Genet.,20(R1):R2-R6; Aucoin et al.,(2006)Biotechnol.Bioeng.95(6):1081-1092; and van Oers et al.,(2015)J.Gen.Virol.96:6-23. Promoters and other such regulatory elements known in the art are explicitly intended for use in nucleic acids of this disclosure. In some embodiments, the promoter is a polyhedral promoter or a p10 promoter. 【0118】 In the example of the first baculovirus vector not encoding the AAV Rep protein, multiple baculovirus vectors, (iii) Further comprising a third baculovirus vector comprising a polynucleotide sequence encoding at least one large AAVRep protein selected from Rep78 and Rep68 and at least one small AAVRep protein selected from Rep52 and Rep40. 【0119】 In this regard, the AAV genome contains Rep genes (i.e., Rep78 and Rep52), and the proteins encoded by these genes function in the replication of the viral genome. Splicing events at the Rep ORF result in the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, unspliced ​​mRNA encoding the Rep78 and Rep52 proteins in insect cells has been shown to be sufficient for the production of AAV vectors. Therefore, in one example, a third baculovirus vector contains a polynucleotide sequence encoding at least one large AAV replication Rep protein selected from Rep78 and Rep68 and at least one small AAVRep protein selected from Rep52 and Rep40. In one example, a third baculovirus vector contains a polynucleotide sequence encoding Rep78 and Rep52. In one example, a third baculovirus vector contains a polynucleotide sequence encoding Rep78 and Rep40. In one example, the third baculovirus vector contains polynucleotide sequences encoding Rep68 and Rep52. In another example, the third baculovirus vector contains polynucleotide sequences encoding Rep68 and Rep40. In yet another example, the third baculovirus vector contains polynucleotide sequences encoding Rep78, Rep68, Rep52, and Rep40. In each of the above examples, the smaller and larger Rep proteins may originate from the same AAV serotype as the viral capsid protein. Alternatively, the smaller and larger Rep proteins may originate from an AAV serotype other than that of the viral capsid protein; for example, the Rep protein may originate from AAV serotype 2. In this regard, Rep sequences are particularly conserved among most serotypes, and Rep sequences have been reported to efficiently cross-complement in insect cells. 【0120】 In each of the above examples describing multiple baculovirus vectors, the polynucleotide sequence encoding the Rep protein in the third baculovirus vector can be operably ligated to a promoter for the expression of the Rep protein in insect cells. Promoters suitable for expression in insect cells are known in the art and are intended for use herein. In one particular example, the promoter may be, for example, a polyhedral promoter or a p10 promoter. Each nucleotide sequence encoding the Rep protein can be operably ligated to the same promoter. Alternatively, each sequence encoding the Rep protein can be operably ligated to its own promoter. 【0121】 At least one of the multiple baculovirus vectors will contain a polynucleotide encoding the assembly activation protein (AAP) required for AAV capsid assembly. For example, a baculovirus vector encoding the capsid protein will contain a polynucleotide encoding the AAP. In another example, a baculovirus encoding the Rep protein and / or a baculovirus encoding the ddRNAi construct and the PABPN1 construct will contain a polynucleotide encoding the AAP. 【0122】 Methods for producing AAVs suitable for use in gene therapy (e.g., non-replicating AAVs) are well known in the art and are intended herein. For example, AAVs can be produced in insect cells using baculovirus systems, as described, for example, US20120028357A1, WO2007046703, US20030148506A1, WO2017184879, US20040197895A1, and WO2007148971 (the contents of which are described herein by reference). Recombinant AAV can also be produced in mammalian cells, both adherent and suspension cells, and the methods for doing so are described in WO2015031686, WO2009097129, WO2007127264, WO1997009441 and WO2001049829, which are described herein by reference. Methods for producing recombinant AAV for use in gene therapy are also described in Berns KI and Giraud C (1996) Biology of adeno-associated virus. Curr Top Microbiol Immunol 218:1-23, Snyder and Flotte (2002) Curr. Opin. Biotechnol., 13:418-423, and Synder RO and Moullier P, Adeno-associated virus; methods and protocols. New York: Humana Press (2011), the contents of which are incorporated herein by reference. 【0123】 ddRNAi construct As described herein, the AAV of this disclosure comprises a DNA-directed RNAi (ddRNAi) construct containing a DNA sequence encoding a short hairpin microRNA (shmiR). The shmiR encoded by the ddRNAi construct is An effector sequence with a length of at least 17 nucleotides; Complementary arrangement of effectors; Stem-loop array; and It contains a primary microRNA (pri-miRNA) backbone. 【0124】 In one example, the effector sequence is substantially complementary to a region of the corresponding length in the RNA transcript described in any one of SEQ ID NOs: 1 to 13. Preferably, the effector sequence is less than 30 nucleotides in length. For example, a preferred effector sequence may be in the range of 17 to 29 nucleotides in length. In a particularly preferred example, the effector sequence is 21 nucleotides in length. More preferably, the effector sequence is 21 nucleotides in length, and the effector complement sequence is 20 nucleotides in length. 【0125】 In certain embodiments, the shmiR encoded by the ddRNAi construct includes an effector sequence that is substantially complementary to a region of the corresponding length in an RNA transcript containing or consisting of any one of SEQ ID NOs: 1-13 (i.e., SEQ ID NOs: 1, 2, 3, 4, 5, 5, 7, 8, 9, 10, 11, 12, or 13). For example, the effector sequence may be substantially complementary to a region of the corresponding length in an RNA transcript containing or consisting of any one of SEQ ID NOs: 1-13 and contain four mismatched bases. For example, the effector sequence may be substantially complementary to a region of the corresponding length in an RNA transcript containing or consisting of any one of SEQ ID NOs: 1-13 and contain three mismatched bases. For example, the effector sequence may be substantially complementary to a region of the corresponding length in an RNA transcript containing or consisting of any one of SEQ ID NOs: 1-13 and contain two mismatched bases. For example, an effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in any one of sequence numbers 1 to 13, and contain one mismatched base. For example, an effector sequence may be 100% complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in any one of sequence numbers 1 to 13. 【0126】 For example, a shmiR encoded by the ddRNAi construct includes an effector sequence that is substantially complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in SEQ ID NO: 9. In this example, the shmiR is also referred to herein as "shmiR13". For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in SEQ ID NO: 9, and contain four mismatched bases. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in SEQ ID NO: 9, and contain three mismatched bases. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in SEQ ID NO: 9, and contain two mismatched bases. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in SEQ ID NO: 9, and contain one mismatched base. For example, the effector sequence may be 100% complementary to a region of the corresponding length in an RNA transcript containing or consisting of the sequence described in Sequence ID No. 9. 【0127】 For example, a shmiR encoded by the ddRNAi construct includes an effector sequence that is substantially complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in SEQ ID NO: 13. In this example, the shmiR is also referred to herein as "shmiR17". For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in SEQ ID NO: 13, and contains four mismatched bases. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in SEQ ID NO: 13, and contains three mismatched bases. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in SEQ ID NO: 13, and contains two mismatched bases. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript containing or consisting of the sequence described in SEQ ID NO: 13, and contains one mismatched base. For example, the effector sequence may be 100% complementary to a region of the corresponding length in an RNA transcript containing or consisting of the sequence described in Sequence ID No. 13. 【0128】 In cases where the effector sequence of shmiR is substantially complementary to a region of corresponding length in the PABPN1 miRNA transcript described herein, and in relation thereto contains 1, 2, 3, or 4 mismatched bases, it is preferable that the mismatch(s) are not located in the region corresponding to the seed region of shmiR, i.e., within nucleotides 2-8 of the effector sequence. 【0129】 In some examples, a ddRNAi construct may include a DNA sequence encoding a shmiR, which includes: (i) an effector sequence that is substantially complementary to the sequence described in SEQ ID NO: 14, except for 1, 2, 3, or 4 base mismatches, and which can form a double helix with the sequence described in SEQ ID NO: 14; and (ii) an effector complement sequence that is substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 15, and an effector complement sequence that is substantially complementary to the sequence described in SEQ ID NO: 15 and can form a double helix with it. The effector complement sequence that is substantially complementary to the sequence described in SEQ ID NO: 15 may be the sequence described in SEQ ID NO: 14. The shmiR in this example will be referred to as "shmiR2" below. 【0130】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR comprising: (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 16, except for 1, 2, 3, or 4 base mismatches, and capable of forming a double helix with the sequence described in SEQ ID NO: 16; and (ii) an effector complement sequence comprising a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 17, and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 17, and capable of forming a double helix with it. The effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 17 may be the sequence described in SEQ ID NO: 16. The shmiR in this example will hereafter be referred to as "shmiR3". 【0131】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR, which includes: (i) an effector sequence that is substantially complementary to the sequence described in SEQ ID NO: 18, except for 1, 2, 3, or 4 base mismatches, and which can form a double helix with the sequence described in SEQ ID NO: 18; and (ii) an effector complement sequence that is substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 19, and an effector complement sequence that is substantially complementary to the sequence described in SEQ ID NO: 19 and can form a double helix with it. The effector complement sequence that is substantially complementary to the sequence described in SEQ ID NO: 19 may be the sequence described in SEQ ID NO: 18. The shmiR in this example will be referred to as "shmiR4" below. 【0132】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR comprising: (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 20, except for 1, 2, 3, or 4 base mismatches, and capable of forming a double helix with the sequence described in SEQ ID NO: 20; and (ii) an effector complement sequence comprising a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 21, and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 21, and capable of forming a double helix with it. The effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 21 may be the sequence described in SEQ ID NO: 20. The shmiR in this example will hereafter be referred to as "shmiR5". 【0133】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR comprising: (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 22, except for 1, 2, 3, or 4 base mismatches, and capable of forming a double helix with the sequence described in SEQ ID NO: 22; and (ii) an effector complement sequence comprising a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 23, and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 23, and capable of forming a double helix with it. The effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 23 may be the sequence described in SEQ ID NO: 22. The shmiR in this example will hereafter be referred to as "shmiR6". 【0134】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR, which includes: (i) an effector sequence that is substantially complementary to the sequence described in SEQ ID NO: 24, except for 1, 2, 3, or 4 base mismatches, and which can form a double helix with the sequence described in SEQ ID NO: 24; and (ii) an effector complement sequence that is substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 25, and an effector complement sequence that is substantially complementary to the sequence described in SEQ ID NO: 25 and can form a double helix with it. The effector complement sequence that is substantially complementary to the sequence described in SEQ ID NO: 25 may be the sequence described in SEQ ID NO: 24. The shmiR in this example will be referred to as "shmiR7" below. 【0135】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR comprising: (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 26, except for 1, 2, 3, or 4 base mismatches, and capable of forming a double helix with the sequence described in SEQ ID NO: 26; and (ii) an effector complement sequence comprising a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 27, and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 27, and capable of forming a double helix with it. The effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 27 may be the sequence described in SEQ ID NO: 26. The shmiR in this example will hereafter be referred to as "shmiR9". 【0136】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR comprising: (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 28, except for 1, 2, 3, or 4 base mismatches, and capable of forming a double helix with the sequence described in SEQ ID NO: 28; and (ii) an effector complement sequence comprising a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 29, and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 29, and capable of forming a double helix with it. The effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 29 may be the sequence described in SEQ ID NO: 28. The shmiR in this example will hereafter be referred to as "shmiR11". 【0137】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR comprising: (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 30, except for 1, 2, 3, or 4 base mismatches, and capable of forming a double helix with the sequence described in SEQ ID NO: 30; and (ii) an effector complement sequence comprising a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 31, and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 31, and capable of forming a double helix with it. The effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 31 may be the sequence described in SEQ ID NO: 30. The shmiR in this example will hereafter be referred to as "shmiR13". 【0138】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR comprising: (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 32, except for 1, 2, 3, or 4 base mismatches, and capable of forming a double helix with the sequence described in SEQ ID NO: 32; and (ii) an effector complement sequence comprising a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 33, and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 33, and capable of forming a double helix with it. The effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 33 may be the sequence described in SEQ ID NO: 32. The shmiR in this example will hereafter be referred to as "shmiR14". 【0139】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR comprising: (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 34, except for 1, 2, 3, or 4 base mismatches, and capable of forming a double helix with the sequence described in SEQ ID NO: 34; and (ii) an effector complement sequence comprising a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 35, and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 35, and capable of forming a double helix with it. The effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 35 may be the sequence described in SEQ ID NO: 34. The shmiR in this example will hereafter be referred to as "shmiR15". 【0140】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR comprising: (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 36, except for 1, 2, 3, or 4 base mismatches, and capable of forming a double helix with the sequence described in SEQ ID NO: 36; and (ii) an effector complement sequence comprising a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 37, and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 37, and capable of forming a double helix with it. The effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 37 may be the sequence described in SEQ ID NO: 36. The shmiR in this example will hereafter be referred to as "shmiR16". 【0141】 For example, a ddRNAi construct may include a DNA sequence encoding a shmiR comprising: (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 38, except for 1, 2, 3, or 4 base mismatches, and capable of forming a double helix with the sequence described in SEQ ID NO: 38; and (ii) an effector complement sequence comprising a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 39, and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 39, and capable of forming a double helix with it. The effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 39 may be the sequence described in SEQ ID NO: 38. The shmiR in this example will hereafter be referred to as "shmiR17". 【0142】 In any of the examples described herein, the shmiR encoded by the ddRNAi construct of this disclosure includes the following in the 5' to 3' direction: 5' adjacent sequence of the pri-miRNA backbone; Complementary arrangement of effectors; Stem-loop array; Effector arrangement; and The 3' adjacent sequence of the pri-miRNA backbone. 【0143】 In any of the examples described herein, the shmiR encoded by the ddRNAi construct of this disclosure includes the following in the 5' to 3' direction: 5' adjacent sequence of the pri-miRNA backbone; Effector arrangement; Stem-loop array; Effector complementary array; and The 3' adjacent sequence of the pri-miRNA backbone. 【0144】 A suitable loop sequence can be selected from those known in the art. However, an exemplary stem-loop sequence is described in Sequence ID No. 40. 【0145】 A primary microRNA (pri-miRNA or pri-R) skeleton suitable for use in the nucleic acids of this disclosure can be selected from those known in the art. For example, the pri-miRNA skeleton can be selected from the pri-miR-30a skeleton, the pri-miR-155 skeleton, the pri-miR-21 skeleton, and the pri-miR-136 skeleton. However, the pri-miRNA skeleton is preferably the pri-miR-30a skeleton. In the example where the pri-miRNA skeleton is the pri-miR-30a skeleton, the 5' adjacent sequence of the pri-miRNA skeleton is described in SEQ ID NO: 41, and the 3' adjacent sequence of the pri-miRNA skeleton is described in SEQ ID NO: 42. Therefore, the ddRNAi constructs encoding the shmiRs of this disclosure (for example, one or more of the shmiR2-shmiR7, shmiR9, shmiR11, and shmiR13-shmiR17 described herein) may include a DNA sequence encoding the sequence described in SEQ ID NO: 41 and a DNA sequence encoding the sequence described in SEQ ID NO: 42. 【0146】 For example, a ddRNAi construct may contain a DNA sequence selected from any one of the sequences described in sequence numbers 56-68. 【0147】 For example, the ddRNAi construct encodes a shmiR (shmiR2) that contains or consists of the DNA sequence described in SEQ ID NO: 56 and contains or consists of the sequence described in SEQ ID NO: 43. 【0148】 For example, the ddRNAi construct contains or comprises the DNA sequence described in SEQ ID NO: 57 and encodes a shmiR (shmiR3) containing or comprising the sequence described in SEQ ID NO: 44. 【0149】 For example, the ddRNAi construct encodes a shmiR (shmiR4) that contains or consists of the DNA sequence described in SEQ ID NO: 58 and contains or consists of the sequence described in SEQ ID NO: 45. 【0150】 For example, the ddRNAi construct encodes a shmiR (shmiR5) that contains or consists of the DNA sequence described in SEQ ID NO: 59 and contains or consists of the sequence described in SEQ ID NO: 46. 【0151】 For example, the ddRNAi construct encodes a shmiR (shmiR6) that contains or consists of the DNA sequence described in SEQ ID NO: 60 and contains or consists of the sequence described in SEQ ID NO: 47. 【0152】 For example, the ddRNAi construct encodes a shmiR (shmiR7) that contains or consists of the DNA sequence described in SEQ ID NO: 61 and contains or consists of the sequence described in SEQ ID NO: 48. 【0153】 For example, the ddRNAi construct contains or comprises the DNA sequence described in SEQ ID NO: 62 and encodes a shmiR (shmiR9) containing or comprising the sequence described in SEQ ID NO: 49. 【0154】 For example, the ddRNAi construct encodes a shmiR (shmiR11) that contains or consists of the DNA sequence described in SEQ ID NO: 63 and contains or consists of the sequence described in SEQ ID NO: 50. 【0155】 For example, the ddRNAi construct encodes a shmiR (shmiR13) that contains or consists of the DNA sequence described in SEQ ID NO: 64 and contains or consists of the sequence described in SEQ ID NO: 51. 【0156】 For example, the ddRNAi construct encodes a shmiR (shmiR14) that contains or consists of the DNA sequence described in SEQ ID NO: 65 and contains or consists of the sequence described in SEQ ID NO: 52. 【0157】 For example, the ddRNAi construct encodes a shmiR (shmiR15) that contains or consists of the DNA sequence described in SEQ ID NO: 66 and contains or consists of the sequence described in SEQ ID NO: 53. 【0158】 For example, the ddRNAi construct encodes a shmiR (shmiR16) that contains or consists of the DNA sequence described in SEQ ID NO: 67 and contains or consists of the sequence described in SEQ ID NO: 54. 【0159】 For example, the ddRNAi construct encodes a shmiR (shmiR17) that contains or consists of the DNA sequence described in SEQ ID NO: 68 and contains or consists of the sequence described in SEQ ID NO: 55. 【0160】 The exemplary ddRNAi constructs of this disclosure encode one or more shmiRs selected from shmiR2, shmiR3, shmiR5, shmiR9, shmiR13, shmiR14, and shmiR17, as described herein. A ddRNAi construct encoding one or more shmiRs selected from shmiR3, shmiR13, shmiR14, and shmiR17, as described herein, is particularly preferred. For example, a ddRNAi construct may encode shmiR13 as described herein. For example, a ddRNAi construct may encode shmiR17 as described herein. 【0161】 Those skilled in the art will understand that the ddRNAi constructs described herein can encode multiple shmiRs that target RNA transcripts corresponding to the PABPN1 protein, which is the causative agent of OPMD. 【0162】 Therefore, in one example, the ddRNAi construct comprises two or more nucleic acids encoding shmiR as described herein, for example, two, three, four, five, six, seven, eight, nine, or ten nucleic acids encoding shmiR as described herein. 【0163】 For example, a ddRNAi construct comprises a nucleic acid containing or comprising a DNA sequence encoding shmiR2, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR2 are described herein and taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 56, a nucleic acid containing or comprising the sequence described in SEQ ID NO: 43, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 56 (shmiR2), and (ii) a nucleic acid containing or comprising a DNA sequence encoding one of shmiR3-shmiR7, shmiR9, shmiR11, or shmiR13-shmiR17. 【0164】 For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR3, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR3 are described herein and shall be taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 57, a nucleic acid comprising or consisting of the sequence described in SEQ ID NO: 44, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 57 (shmiR3), and (ii) a nucleic acid comprising or consisting of a DNA sequence encoding one of shmiR2, shmiR4-shmiR7, shmiR9, shmiR11, or shmiR13-shmiR17. 【0165】 For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR4, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR4 are described herein and shall be taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 58, a nucleic acid comprising or consisting of the sequence described in SEQ ID NO: 45, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 58 (shmiR4), and (ii) a nucleic acid comprising or consisting of a DNA sequence encoding one of shmiR2, shmiR3, shmiR5-shmiR7, shmiR9, shmiR11, or shmiR13-shmiR17. 【0166】 For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR5, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR5 are described herein and shall be taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 59, a nucleic acid comprising or consisting of the sequence described in SEQ ID NO: 46, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 59 (shmiR5), and (ii) a nucleic acid comprising or consisting of a DNA sequence encoding one of shmiR2-shmiR4, shmiR6-shmiR7, shmiR9, shmiR11, or shmiR13-shmiR17. 【0167】 For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR6, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR6 are described herein and taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 60, comprising or consisting of the sequence described in SEQ ID NO: 47, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 60 (shmiR6), and (ii) a nucleic acid comprising or consisting of a DNA sequence encoding one of shmiR2-shmiR5, shmiR7, shmiR9, shmiR11, or shmiR13-shmiR17. 【0168】 For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR7, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR7 are described herein and shall be taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 61, a nucleic acid comprising or consisting of the sequence described in SEQ ID NO: 48, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 61 (shmiR7), and (ii) a nucleic acid comprising or consisting of a DNA sequence encoding one of shmiR2-shmiR6, shmiR9, shmiR11, or shmiR13-shmiR17. 【0169】 For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR9, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR9 are described herein and shall be taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 62, a nucleic acid comprising or consisting of the sequence described in SEQ ID NO: 49, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 62 (shmiR9), and (ii) a nucleic acid comprising or consisting of a DNA sequence encoding one of shmiR2-shmiR7, shmiR11, or shmiR13-shmiR17. 【0170】 For example, a ddRNAi construct comprises a nucleic acid containing or comprising a DNA sequence encoding shmiR11, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR11 are described herein and shall be taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 63, a nucleic acid containing or comprising the sequence described in SEQ ID NO: 50, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 63 (shmiR11), and (ii) a nucleic acid containing or comprising a DNA sequence encoding one of shmiR2-shmiR7, shmiR9, or shmiR13-shmiR17. 【0171】 For example, a ddRNAi construct comprises a nucleic acid containing or comprising a DNA sequence encoding shmiR13, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR13 are described herein and shall be taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 64, a nucleic acid containing or comprising the sequence described in SEQ ID NO: 51, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 64 (shmiR13), and (ii) a nucleic acid containing or comprising a DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11, or shmiR14-shmiR17. 【0172】 For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR14, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR14 are described herein and shall be taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 65, a nucleic acid comprising or consisting of the sequence described in SEQ ID NO: 52, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 65 (shmiR14), and (ii) a nucleic acid comprising or consisting of a DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR13, shmiR15-shmiR17. 【0173】 For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR15, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR15 are described herein and shall be taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 66, comprising or consisting of the sequence described in SEQ ID NO: 53, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 66 (shmiR15), and (ii) a nucleic acid comprising or consisting of a DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11, or shmiR13-shmiR14, shmiR16-shmiR17. 【0174】 For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR16, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR16 are described herein and shall be taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 67, comprising or consisting of the sequence described in SEQ ID NO: 54, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 67 (shmiR16), and (ii) a nucleic acid comprising or consisting of a DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR15, or shmiR17. 【0175】 For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR17, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. Exemplary nucleic acids encoding shmiR17 are described herein and shall be taken for use in the examples of this disclosure. For example, a ddRNAi construct comprises a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 68, a nucleic acid comprising or consisting of the sequence described in SEQ ID NO: 55, and at least one other nucleic acid of this disclosure encoding a shmiR that targets a region of a PABPN1 mRNA transcript. For example, a ddRNAi construct may comprise (i) a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 68 (shmiR17), and (ii) a nucleic acid comprising or consisting of a DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11, or shmiR13-shmiR16. 【0176】 In one example where a ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 1. A suitable nucleic acid encoding a shmiR having an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 1 is described herein, for example, for shmiR2. 【0177】 In one example where a ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 2. A suitable nucleic acid encoding a shmiR having an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 2 is described herein, for example, for shmiR3. 【0178】 In one example where a ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 4. A suitable nucleic acid encoding a shmiR having an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 4 is described herein, for example, for shmiR5. 【0179】 In one example where a ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 7. A suitable nucleic acid encoding a shmiR having an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 7 is described herein, for example, for shmiR9. 【0180】 In one example where a ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 9. A suitable nucleic acid encoding a shmiR having an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 9 is described herein, for example, for shmiR13. 【0181】 In one example where a ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 10. A suitable nucleic acid encoding a shmiR having an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 10 is described herein, for example, for shmiR14. 【0182】 In one example where a ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 13. A suitable nucleic acid encoding a shmiR having an effector sequence substantially complementary to a region of corresponding length in an RNA transcript containing or comprising the sequence described in SEQ ID NO: 13 is described herein, for example, for shmiR17. 【0183】 An exemplary ddRNAi construct encoding multiple shmiRs of the present disclosure comprises at least two nucleic acids, each comprising a DNA sequence encoding a shmiR of the present disclosure, and each shmiR comprising a different effector sequence. 【0184】 In one example, each of at least two nucleic acids may encode a shmiR comprising an effector sequence substantially complementary to a region of corresponding length in the RNA transcript described in one of SEQ ID NOs: 1, 2, 4, 7, 9, 10, and 13. Exemplary nucleic acids of the present disclosure encoding a shmiR comprising an effector sequence substantially complementary to a region of corresponding length in the RNA transcript described in SEQ ID NOs: 1, 2, 4, 7, 9, 10, and 13 are described herein and taken for use in the examples of the present disclosure. 【0185】 For example, a ddRNAi construct includes at least two nucleic acids selected from the following group: A nucleic acid comprising or consisting of a DNA sequence encoding shmiR containing the effector sequence described in SEQ ID NO: 15 and the effector complement sequence described in SEQ ID NO: 14, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 56 (shmiR2); A nucleic acid comprising or consisting of a DNA sequence encoding shmiR containing the effector sequence described in SEQ ID NO: 17 and the effector complement sequence described in SEQ ID NO: 16, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 57 (shmiR3); A nucleic acid comprising or consisting of a DNA sequence encoding shmiR containing the effector sequence described in SEQ ID NO: 21 and the effector complement sequence described in SEQ ID NO: 20, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 59 (shmiR5); A nucleic acid comprising or consisting of a DNA sequence encoding shmiR containing the effector sequence described in SEQ ID NO: 27 and the effector complement sequence described in SEQ ID NO: 26, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 62 (shmiR9); A nucleic acid comprising or consisting of a DNA sequence encoding shmiR containing the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 64 (shmiR13); nucleic acids comprising or consisting thereof a DNA sequence encoding shmiR including the effector sequence described in SEQ ID NO: 33 and the effector complement sequence described in SEQ ID NO: 32, for example, nucleic acids comprising or consisting thereof a DNA sequence described in SEQ ID NO: 65 (shmiR14); and A nucleic acid comprising or consisting of a DNA sequence encoding shmiR containing the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO: 38, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 68 (shmiR17). 【0186】 In one example, each of at least two nucleic acids in the ddRNAi construct encodes a shmiR containing an effector sequence substantially complementary to a region of corresponding length in the RNA transcript described in one of SEQ ID NOs: 2, 9, 10, and 13. Exemplary nucleic acids encoding a shmiR containing an effector sequence substantially complementary to a region of corresponding length in the RNA transcript described in SEQ ID NOs: 2, 9, 10, and 13 are described herein and taken for use in the examples of this disclosure. 【0187】 For example, a ddRNAi construct includes at least two nucleic acids selected from the following group: A nucleic acid comprising or consisting of a DNA sequence encoding shmiR containing the effector sequence described in SEQ ID NO: 17 and the effector complement sequence described in SEQ ID NO: 16, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 57 (shmiR3); A nucleic acid comprising or consisting of a DNA sequence encoding shmiR containing the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 64 (shmiR13); nucleic acids comprising or consisting thereof a DNA sequence encoding shmiR including the effector sequence described in SEQ ID NO: 33 and the effector complement sequence described in SEQ ID NO: 32, for example, nucleic acids comprising or consisting thereof a DNA sequence described in SEQ ID NO: 65 (shmiR14); and A nucleic acid comprising or consisting of a DNA sequence encoding shmiR containing the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO: 38, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 68 (shmiR17). 【0188】 For example, a ddRNAi construct includes a nucleic acid encoding a shmiR containing an effector sequence substantially complementary to a region of corresponding length in the RNA transcript described in SEQ ID NO: 9, and a nucleic acid encoding a shmiR containing an effector sequence substantially complementary to a region of corresponding length in the RNA transcript described in SEQ ID NO: 13. For example, a ddRNAi construct may include: (a) a nucleic acid comprising or consisting of a DNA sequence encoding shmiR including the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 64 (shmiR13); and (b) A nucleic acid comprising or consisting of a DNA sequence encoding shmiR including the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO: 38, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 68 (shmiR17). 【0189】 The exemplary ddRNAi constructs of this disclosure include nucleic acids comprising or consisting of the DNA sequence described in SEQ ID NO: 64 (shmiR13) and nucleic acids comprising or consisting of the DNA sequence described in SEQ ID NO: 68 (shmiR17). 【0190】 For example, a ddRNAi construct includes a nucleic acid encoding a shmiR containing an effector sequence substantially complementary to a region of corresponding length in the RNA transcript described in SEQ ID NO: 2, and a nucleic acid encoding a shmiR containing an effector sequence substantially complementary to a region of corresponding length in the RNA transcript described in SEQ ID NO: 10. For example, a ddRNAi construct may include: (a) a nucleic acid comprising or consisting of a DNA sequence encoding shmiR including the effector sequence described in SEQ ID NO: 17 and the effector complement sequence described in SEQ ID NO: 16, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 57 (shmiR3); and (b) A nucleic acid comprising or consisting of a DNA sequence encoding shmiR including the effector sequence described in SEQ ID NO: 33 and the effector complement sequence described in SEQ ID NO: 32, for example, a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 65 (shmiR14). 【0191】 The exemplary ddRNAi constructs of this disclosure include nucleic acids comprising or consisting of the DNA sequence described in SEQ ID NO: 57 (shmiR3), and nucleic acids comprising or consisting of the DNA sequence described in SEQ ID NO: 65 (shmiR14). 【0192】 In an example of a ddRNAi construct encoding two or more shmiRs, two or more of the nucleic acids encoding the shmiRs may form distinct portions of the same polynucleotide within the ddRNAi construct. 【0193】 In some examples, the nucleic acid encoding a shmiR, or each nucleic acid, may include or be operably ligated to additional elements, for example, to facilitate the transcription of a shmiR. For example, a ddRNAi construct may include one or more promoters operably ligated to the sequence(s) encoding the shmiR(s) described herein. Other elements, such as transcription terminators and initiators, are known in the art and / or described herein. 【0194】 In each of the aforementioned examples describing the ddRNAi constructs of this disclosure, the nucleic acid encoding a shmiR, or each nucleic acid, may be operably ligated to a promoter. For example, the ddRNAi construct described herein may include, for example, a single promoter operably ligated to the nucleic acid encoding a shmiR contained therein, or each nucleic acid, in order to drive the expression of one or more shmiRs from the ddRNAi construct. In another example, each nucleic acid encoding a shmiR contained in the ddRNAi construct is operably ligated to a separate promoter. 【0195】 In an example where multiple promoters exist, the promoters may be the same or different. For example, a construct may contain multiple copies of the same promoter, each copy operably ligated to a different nucleic acid of the Disclosure. In another example, each promoter operably ligated to a nucleic acid encoding a shmiR of the Disclosure may be different. For example, in a ddRNAi construct encoding two shmiRs, the two nucleic acids encoding the shmiRs are each operably ligated to a different promoter. 【0196】 In one example, the promoter is a constitutive promoter. The term "constitutive," when applied to a promoter, means that the promoter can direct the transcription of an operablely linked nucleic acid sequence in the absence of a specific stimulus (e.g., heat shock, chemical, light, etc.). Typically, a constitutive promoter can direct the expression of a coding sequence in substantially any cell and any tissue. Promoter examples used for shmiR transcription include ubiquitin, CMV, β-actin, histone H4, EF-1α, the pgk gene promoter regulated by RNA polymerase II, or a promoter element regulated by RNA polymerase I. 【0197】 For example, Pol II promoters such as CMV, SV40, U1, β-actin, or hybrid Pol II promoters may be used. Other suitable Pol II promoters are known in the art and may be used in accordance with this example of the disclosure. For example, Pol II promoter systems may be preferred within the ddRNAi constructs of the disclosure that express pri-miRNAs which are processed into one or more shmiRs by the action of the enzymes Drosha and Pasha. Pol II promoter systems may also be preferred within the ddRNAi constructs of the disclosure that include sequences encoding multiple shmiRs under the control of a single promoter. Pol II promoter systems may also be preferred when tissue specificity is desired. 【0198】 In other examples, promoters controlled by RNA polymerase III may be used, such as the U6 promoter (U6-1, U6-8, U6-9), the H1 promoter, the 7SL promoter, the human Y promoter (hY1, hY3, hY4 (see Marai, et al., Nucleic Acids Res 22(15):3045-52 (1994)) and hY5 (see Marai, et al., Nucleic Acids Res 24(18):3552-59 (1994))), the human MRP-7-2 promoter, the adenovirus VA1 promoter, the human tRNA promoter, or the 5s ribosomal RNA promoter. 【0199】 Promoters suitable for use within the ddRNAi construct of this disclosure are described in U.S. Patent Nos. 8,008,468 and U.S. Patent Nos. 8,129,510. 【0200】 In one example, the promoter is the RNA pol III promoter. For example, the promoter is the U6 promoter (e.g., U6-1, U6-8, or U6-9 promoter). In another example, the promoter is the H1 promoter. 【0201】 In the case of a ddRNAi construct of this disclosure encoding multiple shmiRs, each nucleic acid in the ddRNAi construct can be operably linked to a U6 promoter, for example, a separate U6 promoter. 【0202】 For example, the promoter of the ddRNAi construct is the U6 promoter. For example, the promoter could be the U6-1 promoter. For example, the promoter could be the U6-8 promoter. For example, the promoter could be the U6-9 promoter. 【0203】 In some cases, promoters of varying strengths are used. For example, the use of two or more strong promoters (such as a Pol III type promoter) can burden the cell, for instance, by depleting the pool of available nucleotides or other cellular components necessary for transcription. Additionally or alternatively, the use of several strong promoters can lead to toxic levels of shmiR expression in the cell. Thus, in some cases, one or more promoters in a multi-promoter ddRNAi construct may be weaker than the others in the construct, or all promoters in the construct may express shmiR at a rate below maximum. Promoters can also be modified to achieve weaker or stronger levels of transcription using various molecular techniques or in other ways, for example, by modifying various regulatory elements. One means of achieving reduced transcription is to modify sequence elements within the promoter that are known to control promoter activity. For example, proximal sequence elements (PSEs) are known to affect the activity of the human U6 promoter (see Domitrovich, et al., Nucleic Acids Res 31:2344-2352 (2003)). Replacing PSE elements present in strong promoters such as the human U6-1, U6-8, and U6-9 promoters with elements from weak promoters such as the human U6-7 promoter reduces the activity of hybrid U6-1, U6-8, or U6-9 promoters. This approach is used in the examples described in this application, but other means for achieving this result are known in the art. 【0204】 The promoters useful in the ddRNAi constructs of this disclosure may also be tissue-specific or cell-specific. The term “tissue-specific” as applied to a promoter refers to a promoter that can direct the selective transcription of a target nucleic acid to a specific type of tissue (e.g., eye or muscle tissue) when the expression of the same nucleotide sequence of interest is relatively absent in different tissue types (e.g., liver). The term “cell-specific” as applied to a promoter refers to a promoter that can direct the selective transcription of a target nucleic acid to a specific type of cell when the expression of the same nucleotide sequence of interest is relatively absent in different types of cells within the same tissue. For example, muscle-specific promoters such as Spc512 or CK8 are used. However, other muscle-specific promoters are known in the art and are intended for use in combination with the ddRNAi constructs of this disclosure. 【0205】 For example, the ddRNAi construct of this disclosure may further include one or more enhancers for increasing the expression of shmiR as described herein. Suitable enhancers for use in the examples of this disclosure include the Apo E HCR enhancer, the CMV enhancer (Xia et al, Nucleic Acids Res 31-17 (2003)), and other enhancers known to those skilled in the art. Suitable enhancers for use in the ddRNAi construct of this disclosure are described in U.S. Patent No. 8,008,468. 【0206】 In further examples, the ddRNAi constructs of this disclosure may include transcriptional terminators ligated to nucleic acids encoding the shmiRs of this disclosure. In the case of a ddRNAi construct that includes multiple nucleic acids as described herein, i.e., encoding multiple shmiRs, the terminators ligated to each nucleic acid may be identical or different. For example, in the ddRNAi constructs of this disclosure using the RNA pol III promoter, the terminator may be a contiguous stretch of four or more, five or more, or six or more T residues. However, if different promoters are used, the terminators may be different and match the promoter from which the terminator originates. Such terminators include, but are not limited to, the SV40 polyA, AdV VA1 gene, 5S ribosomal RNA gene, and human tRNA terminators. Other promoter and terminator combinations are known in the art and are intended for use in the ddRNAi constructs of this disclosure. 【0207】 Furthermore, promoters and terminators can be mixed and matched, as is commonly done with RNA pol II promoters and terminators. 【0208】 For example, the combination of promoters and terminators used for each nucleic acid in a ddRNAi construct containing multiple nucleic acids differs to reduce the likelihood of DNA recombination events occurring between components. 【0209】 One exemplary ddRNAi construct of this disclosure comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR13 as described herein, operably ligated to a promoter, and a nucleic acid comprising or consisting of a DNA sequence encoding shmiR17 as described herein, operably ligated to a promoter. For example, an exemplary ddRNAi construct of this disclosure comprises a nucleic acid comprising or consisting of a DNA sequence described in SEQ ID NO: 64, operably ligated to a promoter, and a nucleic acid comprising or consisting of a DNA sequence described in SEQ ID NO: 68, operably ligated to a promoter. In one example, each nucleic acid in a ddRNAi construct encoding shmiR is operably ligated to a separate promoter. In another example, each nucleic acid in a ddRNAi construct encoding shmiR is operably ligated to the same promoter. For example, the promoter or each promoter may be a U6 promoter, e.g., U6-1, U6-8, or U6-9 promoter. For example, the promoter or each promoter may be a muscle-specific promoter, e.g., Spc512 or CK8 promoter. 【0210】 In one example, where the nucleic acids in the ddRNAi construct encoding shmiR13 and shmiR17 are operably linked to the same Spc512 promoter, the ddRNAi construct contains or comprises the DNA sequence described in SEQ ID NO: 72. In another example, where the nucleic acids in the ddRNAi construct encoding shmiR13 and shmiR17 are operably linked to the same CK8 promoter, the ddRNAi construct contains or comprises the DNA sequence described in SEQ ID NO: 70. 【0211】 One exemplary ddRNAi construct of this disclosure comprises a nucleic acid comprising or consisting of a DNA sequence encoding shmiR3 as described herein, operably ligated to a promoter, and a nucleic acid comprising or consisting of a DNA sequence encoding shmiR14 as described herein, operably ligated to a promoter. For example, an exemplary ddRNAi construct of this disclosure comprises a nucleic acid comprising or consisting of a DNA sequence described in SEQ ID NO: 57, operably ligated to a promoter, and a nucleic acid comprising or consisting of a DNA sequence described in SEQ ID NO: 65, operably ligated to a promoter. In one example, each nucleic acid in a ddRNAi construct encoding shmiR is operably ligated to a separate promoter. In another example, each nucleic acid in a ddRNAi construct encoding shmiR is operably ligated to the same promoter. For example, the promoter or each promoter may be a U6 promoter, e.g., U6-1, U6-8, or U6-9 promoter. For example, the promoter or each promoter may be a muscle-specific promoter, e.g., Spc512 or CK8 promoter. 【0212】 In the example where the nucleic acids in the ddRNAi construct encoding shmiR3 and shmiR14 are operably linked to the same Spc512 promoter, the ddRNAi construct contains or comprises the DNA sequence described in SEQ ID NO: 71. In the example where the nucleic acids in the ddRNAi construct encoding shmiR3 and shmiR14 are operably linked to the same CK8 promoter, the ddRNAi construct contains or comprises the DNA sequence described in SEQ ID NO: 69. 【0213】 Furthermore, the ddRNAi construct can include one or more multiple cloning sites and / or unique restriction sites strategically placed such that the promoter, nucleic acid encoding the shmiR, and / or other regulatory elements can be easily removed or replaced. The ddRNAi construct can be assembled from smaller oligonucleotide components using strategically placed restriction sites and / or complementary sticky ends. A basic vector for one approach according to the present disclosure includes a plasmid having a multiple linker where all sites are unique (this is not an absolute requirement). Subsequently, each promoter is inserted between its designated unique sites to form a base cassette having one or more promoters, all of which can have variable orientations. Subsequently, again, annealed primer pairs are inserted into unique sites downstream of each of the individual promoters, resulting in single, double, or multiple expression cassette constructs. The insert can be moved into the AAV backbone using two unique restriction enzyme sites (same or different sites) adjacent to the single-expression, double-expression, or multiple-expression cassette insert. 【0214】 The production of ddRNAi constructs can be achieved using any suitable genetic engineering techniques known in the art, including but not limited to standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing. The ddRNAi construct (or polynucleotide containing the same) may also include sequences necessary for packaging the ddRNAi construct into viral particles and / or sequences that enable integration of the ddRNAi construct into the genome of the target cell. In some examples, or each viral construct, further includes genes that enable viral replication and dissemination, although such genes are provided in trans. Additionally, the viral construct or each viral construct includes a gene or gene sequence from the genome of any known organism, either incorporated in its native form or modified. For example, the viral construct may include sequences useful for replication of the construct in bacteria. 【0215】 Testing of the shmiR or ddRNAi constructs of this disclosure Cell culture model An example of a cell line useful as a cell culture model of OPMD is the HEK293T cell line (HEK293T, ATCC, Manassas, USA), which has been transfected with a vector expressing normal Ala10-human PABPN1-FLAG (Ala10) or mutant Ala17-human PABPN1-FLAG (Ala17), the latter being characteristic of OPMD. 【0216】 Further examples of cell lines useful as cell culture models of OPMD are C2C12 mouse muscle cells and ARPE-19 human retinal cells. 【0217】 Another example of a cell line useful as a cell culture model for OPMD is a primary mouse myoblast (IM2) cell line stably transfected to express either normal Ala10-human PABPN1-FLAG (Ala10) or mutant Ala17-human PABPN1-FLAG (Ala17). An exemplary IM2-derived cell line that stably expresses mutant Ala17-human PABPN1-FLAG (Ala17) is the H2kB-D7e cell line. The H2kB-D7e cell line is also described in Raz et al., (2011) American Journal of Pathology, 179(4):1988-2000. 【0218】 Other cell lines suitable as cell culture models for OPMD are known in the art, including those described in Fan et al., (2001) Human Molecular Genetics, 10:2341-2351, Bao et al., (2002) The Journal of Biological Chemistry, 277:12263-12269, and Abu-Baker et al., (2003) Human Molecular Genetics, 12:2609-2623. 【0219】 As illustrated herein, the activity of the shmiR disclosed herein is determined by administering a nucleic acid encoding shmiR, or a ddRNAi construct or expression vector containing it, to cells, and then measuring the expression level of RNA or protein encoded by the PABPN1 gene. For example, intracellular PABPN1 gene expression can be assayed using one or more probes or primers specific to PABPN1 by one or more of the following: RT-PCR, quantitative PCR, semi-quantitative PCR, or in situ hybridization under stringent conditions. PABPN1 mRNA or DNA can also be assayed by PCR using one or more probes or primers specific to PABPN1, and the PABPN1 protein can also be detected using Western blotting or ELISA. 【0220】 Polynucleotides that can be used in RT-PCR, quantitative PCR, or semi-quantitative PCR techniques for detecting PABPN1 expression are known and commercially available (Thermo Fisher). However, polynucleotides useful for PCR-based detection methods can be designed based on available sequence information for PABPN1 using methods and / or software known in the art. For example, the presence or absence of PABPN1 mRNA can be detected using RT-PCR using standard methodologies known in the art. For example, the presence or absence or relative amount of PABPN1 polypeptide or protein can be detected using one or more of the following: Western blotting, ELISA, or other standard quantitative or semi-quantitative techniques available in the art, or a combination of such techniques. Techniques that rely on antibody recognition of PABPN1 are intended and described herein. For example, the presence or absence or relative amount of PABPN1 polypeptide can be detected by a technique that includes antibody capture of PABPN1 polypeptide combined with electrophoretic degradation of the captured PABPN1 polypeptide, for example, using the Isonostic® assay (Target Discovery, Inc.). Antibodies against the PABPN1 protein are commercially available. 【0221】 Various methods for normalizing the difference between transfection or transduction efficiency and sample recovery are known in the art. 【0222】 Nucleic acids, ddRNAi constructs, or expression vectors of the disclosed herein that reduce the expression of mRNA or protein encoded by PABPN1, or the presence of PABPN1 protein nuclear aggregates, compared to the level of mRNA expression or protein or the amount of PABPN1 protein nuclear aggregates in the absence of the RNA of the disclosed herein, are considered useful for therapeutic applications, such as treating OPMD by reducing the expression of endogenous PABPN1 and replacing some or all of the endogenous PABPN1 with PABPN1 proteins that are not the cause of OPMD as described herein. 【0223】 Animal models Several small animal models are available for use in OPMD research, as described in Uyama et al., (2005) Acta Myologica, 24(2):84-88 and Chartier and Simonelig (2013) Drug Discovery Today:technologies, 10:e103-107. An exemplary animal model is the A17.1 transgenic mouse model previously described in Davies et al., (2005) Nature Medicine, 11:672-677 and Trollet et al., (2010) Human Molecular Genetics, 19(11):2191-2207. 【0224】 The effectiveness of the shmiR or ddRNAi constructs of this disclosure for knocking down, reducing, or inhibiting the expression of RNA or protein encoded by the PABPN1 gene can be determined using any of the aforementioned animal models. 【0225】 Methods for assaying PABPN1 gene expression are described herein with respect to cell models and should be applied mutatis mutandis to the examples in this disclosure. 【0226】 PABPN1 construct As described herein, the AAVs of this disclosure comprise a polynucleotide sequence comprising the PABPN1 construct. In this regard, the AAVs of this disclosure provide, for example, agents for the replacement of a functional PABPN1 protein in cells or animals. The functional PABPN1 protein is not the cause of OPMD, nor is it encoded by an mRNA transcript targeted by shmiR(s) encoded by the ddRNAi construct described herein contained within the AAV. 【0227】 For example, the PABPN1 construct includes a nucleic acid, such as DNA or cDNA, that encodes a functional PABPN1 protein. For example, the nucleic acid encoding the functional PABPN1 protein may be codon-optimized. For example, it may contain one or more degenerate or fluctuating bases relative to the wild-type PABPN1 nucleic acid, but encode the same amino acids, so the corresponding mRNA sequence encoding the functional PABPN1 protein is not recognized by the shmiR(s) encoded and expressed from the ddRNAi construct. For example, the codon-optimized nucleic acid encoding the functional PABPN1 protein may contain one or more degenerate or fluctuating bases relative to the wild-type PABPN1 nucleic acid in a region targeted by one or more shmiR(s) encoded and expressed from the ddRNAi construct. For example, the one or more degenerate or fluctuating bases are located within the seed region of the effector sequence of the shmiR(s) encoded and expressed from the ddRNAi construct. 【0228】 In one example, a nucleic acid having a PABPN1 construct encoding a functional PABPN1 protein is codon-optimized such that its corresponding mRNA sequence is not recognized by shmiR(s) encoded and expressed from a ddRNAi construct. Preferably, the functional PABPN1 protein encoded by the codon-optimized nucleic acid sequence contains the amino acid sequence described in SEQ ID NO: 74, i.e., the amino acid sequence of the wild-type human PABPN1 protein. Those skilled in the art will understand that there are multiple combinations of nucleotide sequences that can be used to encode a functional PABPN1 protein, and the selection of the nucleotide sequence ultimately depends on the effector sequences of shmiR(s) encoded and expressed from a ddRNAi construct. That is, the codon-optimized nucleic acid will not be recognized by shmiR(s). In one example, the PABPN1 construct contains a nucleic acid containing the sequence described in SEQ ID NO: 73. In one example, the nucleic acid encoding a functional PABPN1 protein may also contain a Kozak sequence. 【0229】 In one example, a codon-optimized nucleic acid encoding a functional PABPN1 protein is operably ligated to a promoter suitable for the expression of the functional PABPN1 protein. A promoter suitable for the expression of the functional PABPN1 protein in muscle may be particularly preferred. One exemplary promoter suitable for use with a nucleic acid encoding a functional PABPN1 protein is the Spc512 promoter. Another exemplary promoter suitable for use with a nucleic acid encoding a functional PABPN1 protein is the CK8 promoter. However, any suitable promoter known in the art may be used. For example, other suitable promoters for use with nucleic acids encoding a functional PABPN1 protein are described in US20110212529A1. 【0230】 In one example, the PABPN1 construct and the ddRNAi construct are operably linked to the same promoter within the same polynucleotide, for example, they are both operably linked to the Spc512 promoter. According to this example, a single promoter drives the expression of the functional PABPN1 protein and the shmiR. 【0231】 As described herein, the promoters useful in some examples of the present disclosure can be tissue-specific or cell-specific. 【0232】 In one example, the codon-optimized nucleic acid encoding the functional PABPN1 protein of the present disclosure may further include one or more enhancers for increasing the expression of the functional PABPN1 protein and its corresponding mRNA transcript. Enhancers suitable for use in this example of the present disclosure will be known to those skilled in the art. 【0233】 Functional PABPN1 testing Animal models Exemplary animal models for studying OPMD are described. 【0234】 Using any of the aforementioned animal models, the efficacy of a PABPN1 construct or an AAV containing it for replacing the functional PABPN1 protein in vivo in the presence of one or more shmiRs expressed from the ddRNAi of the present disclosure can be determined. 【0235】 Methods for assaying PABPN1 expression are described herein with respect to cell models and are to be taken as applicable to this example of the present disclosure. 【0236】 For example, histological and morphological analyses can be used to determine the efficacy of the agent of the disclosure for in vivo substitution of the functional PABPN1 protein in the presence of one or more shmiRs expressed from the ddRNAi of the disclosure. Further assays that can be used to determine the efficacy of the agent of the disclosure for in vivo substitution of the functional PABPN1 protein are described in Trollet et al., (2010) Human Molecular Genetics, 19(11):2191-2207. 【0237】 PABPN1 "Silence and Substitution" DNA Construct As described herein, the AAVs of this disclosure comprise a single polynucleotide comprising the ddRNAi construct and the PABPN1 construct as described herein. That is, the ddRNAi construct and the PABPN1 construct may be provided as a combined DNA construct (also referred herein as the “silence and substitution” construct or SR construct), which is packaged in a modified AAV as described herein for delivery to a patient. An exemplary DNA construct comprising the nucleic acid encoding a functional PABPN1 protein and the ddRNAi construct of this disclosure is described in Example 2. 【0238】 A single DNA construct comprising a ddRNAi construct and a PABPN1 construct may, for example, include one or more promoters and / or shmiRs encoded by the ddRNAi construct to drive the expression of a functional PABPN1 protein. In some examples of this disclosure, useful promoters may be tissue-specific or cell-specific. Exemplary promoters are, for example, muscle-specific promoters such as Spc512 and CK8. However, any suitable promoter known in the art, such as those described in US20110212529A1, is intended for use within the DNA constructs described herein. 【0239】 The DNA constructs, including the ddRNAi construct and the PABPN1 construct, are packaged in modified AAVs as described herein for delivery to patients. 【0240】 In one example, the DNA construct comprises, from 5' to 3', a muscle-specific promoter, e.g., the Spc512 promoter, the PABPN1 construct as described herein, and the ddRNAi construct as described herein, where the ddRNAi construct is located within the 3' untranslated region (UTR) of the nucleic acid encoding the functional PABPN1 protein. The DNA construct according to this example is shown in Figure 1A. 【0241】 The exemplary DNA construct in this embodiment includes the following in the 5' to 3' direction: (a) Muscle-specific promoter, e.g., Spc512; (b) The PABPN1 construct described herein, comprising a DNA sequence encoding a functional PABPN1 protein having an mRNA transcript that is not targeted by shmiR encoded by the ddRNAi construct; (c) A ddRNAi construct of this disclosure comprising a nucleic acid comprising a DNA sequence encoding shmiR17 as described herein and a nucleic acid comprising a DNA sequence encoding shmiR13 as described herein. 【0242】 According to this embodiment, the DNA construct may include or consist of the DNA sequence described in Sequence ID No. 72. 【0243】 Exemplary ddRNAi constructs encoding shmiR13 and shmiR17 for inclusion in the DNA constructs of this disclosure include a nucleic acid comprising or comprising a DNA sequence encoding a shmiR that includes the effector sequence described in SEQ ID NO: 31 and an effector complement sequence substantially complementary to the sequence described in SEQ ID NO: 31, for example, the effector complement sequence described in SEQ ID NO: 30 (shmiR13), and an effector complement sequence comprising or comprising an effector complement sequence that is substantially complementary to the sequence described in SEQ ID NO: 39, for example, an effector complement sequence that is substantially complementary to the sequence of SEQ ID NO: 38 (shmiR17). For example, a ddRNAi construct by example of a DNA construct may include a ddRNAi construct comprising a nucleic acid comprising or comprising the DNA sequence described in SEQ ID NO: 64 (shmiR13), and a nucleic acid comprising or comprising the DNA sequence described in SEQ ID NO: 68 (shmiR17). 【0244】 An exemplary PABPN1 construct for inclusion within the DNA construct of this disclosure comprises the codon-optimized sequence described in SEQ ID NO: 73 and encodes the functional PABPN1 protein described in SEQ ID NO: 74. 【0245】 While specific examples are provided, it will be understood that the DNA constructs according to this disclosure may include any ddRNAi constructs described herein that encode one or more shmiRs that target the RNA transcript of PABPN1. However, the ddRNAi constructs encoding the shmiRs described in Examples 1 to 5 herein may be particularly suitable for inclusion within the DNA constructs of this disclosure. Similarly, it will be understood that the DNA constructs according to this disclosure may include any PABPN1 construct that encodes a functional PABPN1 protein, the transcript of which is not targeted by the shmiR expressed from the ddRNAi construct. 【0246】 Compositions and carriers In some cases, the AAVs of this disclosure may be provided within a pharmaceutical composition formulated for delivery to a patient, for example, a human patient. 【0247】 The compositions of this disclosure may also comprise one or more pharmaceutically acceptable carriers or diluents. For example, a composition may comprise a carrier suitable for delivery to the target muscle after administration of the AAV of this disclosure to that muscle. Carriers suitable for formulation and delivery of AAV are known in the art and are intended herein. 【0248】 The compositions will preferably include materials that increase the biological stability of the AAVs of this disclosure and / or materials that increase the ability of the AAVs to selectively localize to and / or penetrate muscle cells. The therapeutic compositions of this disclosure may be administered with a pharmaceutically acceptable carrier (e.g., saline) selected based on the mode and route of administration and standard pharmaceutical practice. Those skilled in the art can readily formulate pharmaceutical compositions comprising one or more AAVs of this disclosure. In some cases, isotonic formulations are used. Generally, additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Stabilizers include gelatin and albumin. In some examples, vasoconstrictors are added to the formulations. The compositions according to this disclosure are provided sterile and pyrogen-free. The requirements for suitable pharmaceutical carriers and pharmaceuticals for use in pharmaceutical formulations are described in Remington: The Science and Practice of Pharmacy (formerly Remington's Pharmaceutical Sciences), Mack Publishing Co., the standard reference text in this field, and the USP / NF). 【0249】 The volume, concentration, and formulation of the pharmaceutical composition, as well as the administration regimen, can be specifically adjusted to maximize cell delivery while minimizing toxicity such as inflammatory responses. For example, relatively large volumes (5, 10, 20, 50 ml or more) containing the corresponding low concentration of the active ingredient, as well as those containing anti-inflammatory compounds such as corticosteroids, may be available, if desired. 【0250】 The compositions of this disclosure may be formulated for administration by any preferred route (e.g., preferred for delivery to the pharyngeal muscles of a target). For example, routes of administration include, but are not limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intra-arterial, intraocular, and oral, as well as transdermal, inhalation, or suppository. Exemplary routes of administration include intravenous (IV), intramuscular (IM), oral, intraperitoneal, intradermal, intra-arterial, and subcutaneous injection. In one example, the compositions of this disclosure are formulated for IM administration (e.g., formulated for administration to the pharyngeal muscles). In a preferred embodiment, administration is carried out directly to the pharyngeal muscles of a target. For example, the pharyngeal muscles may include one or more of the inferior pharyngeal muscles, middle pharyngeal muscles, superior pharyngeal constrictor muscles, palatinaryngeal muscles, tubal pharyngeal muscles, stylopharyngeal muscles, or any combination thereof. In another preferred embodiment, administration is carried out directly to the muscles of the tongue of a target. Such compositions are useful for pharmaceutical applications and can be readily incorporated into suitable sterile, non-pyrogenic vehicles, such as buffered saline for injection, for parenteral administration, e.g., IM (e.g., directly into the pharyngeal muscle), intravenous administration (e.g., intravenous infusion), SC, and intraperitoneal administration. In preferred embodiments, administration routes such as IM (e.g., directly into the pharyngeal muscle) achieve effective delivery to muscle tissue and transduction of the ddRNAi construct and codon-optimized nucleic acid encoding PABPN1 of the Disclosure, as well as expression of shmiR and the codon-optimized nucleic acid therein. 【0251】 Treatment method A particular aspect of this disclosure relates to administering to a human subject in need of it an AAV or a composition comprising AAV as described herein to treat the subject, and inhibiting the expression of endogenous PABPN1 proteins, such as the PABPN1 protein that causes OPMD, in the subject, wherein the composition is administered by direct injection into the pharyngeal muscle of the subject. 【0252】 In some embodiments, AAV or compositions containing AAV described herein can be used to treat OPMD in subjects suffering from OPMD. Similarly, AAV or compositions containing AAV described herein can be used to prevent the onset or progression of one or more symptoms of OPMD in subjects suffering from or predisposed to OPMD. 【0253】 In some embodiments, subjects improved their swallowing after administration of the AAV or AAV-containing composition described herein by direct injection into the pharyngeal muscles of the subjects. 【0254】 As described herein, the AAV and / or compositions of the Disclosure comprise both the ddRNAi construct of the Disclosure and the PABPN1 construct of the Disclosure comprising a codon-optimized nucleic acid encoding the functional PABPN1 protein of the Disclosure. Therefore, administration of the AAV or composition may effectively (i) inhibit, reduce, or knock down the expression of endogenous PABPN1, such as the PABPN1 protein containing the expanded polyalanine tube that causes OPMD, and (ii) result in the expression of a functional PABPN1 protein that is not targeted by shmiR that inhibits, reduces, or knocks down the expression of endogenous PABPN1. Thus, the AAV or composition of the Disclosure may restore the function of the PABPN1 protein, for example, post-transcriptional processing of RNA, in the cells or animals to which it is administered. 【0255】 In certain embodiments, treatment of OPMD may include administration of an AAV or a composition containing an AAV as described herein by direct injection into the pharyngeal muscle of the subject. 【0256】 In some embodiments, the administration route is IM (e.g., direct injection into the pharyngeal muscle of the subject), which achieves effective delivery to muscle tissue and transduction of the ddRNAi construct and PABPN1 construct of this disclosure, which include codon-optimized nucleic acid encoding PABPN1, and expression of shmiR targeting wild-type PABPN1 mRNA transcript, and expression of codon-optimized nucleic acid within it. 【0257】 The therapeutically effective dose level for any particular patient depends on a variety of factors, including the patient's age, weight, overall health, sex, diet; administration time; route of administration; sequence of administration of the AAV or AAV-containing composition described herein; duration of treatment; and other relevant factors. 【0258】 The efficacy of the AAV or compositions comprising the AAV of this disclosure for reducing or inhibiting the expression of the PABPN1 protein that causes OPMD and for expressing a functional PABPN1 protein that does not cause OPMD in an amount sufficient to restore PABPN1 function may be determined by evaluating the muscle contraction characteristics and / or dysphagia of the subject of treatment. Methods for testing swallowing ability and muscle contraction characteristics are known in the art. For example, dysphagia may be evaluated using videofluoroscopy, UGI endoscopy, or esophagoscopy and impedance testing. Other methods for evaluating the clinical features of OPMD are described in Ruegg et al, (2005) Swiss Medical Weekly, 135:574-586. 【0259】 [Table 1] 【0260】 [Table 2] 【0261】 [Table 3] 【0262】 [Table 4] [Examples] 【0263】 [Example 1 - Design of shmiR targeting PABPN1] Sequences representing potential targets for siRNA construct design were identified from PABPN1 mRNA sequences using publicly available siRNA design algorithms (Ambion, Promega, Invitrogen, Origene, MWG, etc.). The selected sequences were conserved within human, non-human primate, bovine, and mouse species. Sequences encoding candidate siRNAs were incorporated into a pre-miR30a scaffold to construct sequences encoding short hairpin microRNAs (shmiRs) containing the 5' facile region (SEQ ID NO: 41), siRNA sense strand sequence (effector complement sequence), stem / loop junction sequence (SEQ ID NO: 40), siRNA antisense strand (effector sequence), and 3' facile region (SEQ ID NO: 42). The predicted secondary structures of representative shmiRs are shown in Figure 1C. The target regions of the PABPN1 mRNA transcripts of the designed shmiRs are shown in Table 1, and the corresponding shmiR effector sequences (antisense strands) are shown in Table 2. 【0264】 [Example 2 - Generation of a single "silence and substitution construct" for simultaneous gene silencing of endogenous PABPN1 and substitution with codon-optimized PABPN1] We constructed single-stranded adeno-associated virus type 2 (ssAAV2) plasmids expressing shmiR17 and shmiR13 (e.g., as listed in Tables 3 and 4) in combination with the optPABPN1 sequence. 【0265】 Silence and substitution constructs (hereinafter referred to as "SR constructs") were generated by subcloning the DNA sequences encoding shmiR17 and shmiR13 (listed in Table 4) into the 3' untranslated region of the optPABPN1 transcript within the pAAV2 vector backbone (pAAV-shmiR viral plasmid). Expression of both optPABPN1 and the two shmiRs in a single transcript is driven by the muscle-specific promoter Spc512. Schematic diagrams of the SR constructs are shown in Figures 1(A), 1(B), and 2. 【0266】 Next, recombinant pseudo-AAV vector stocks were generated. Briefly, HEK293T cells were cultured in Dulbecco's Modified Eagle Medium in a cell factory, supplemented with 10% FBS, at 37°C and 5% CO2. The pAAV-shmiR viral plasmid (SR-construct) was complexed with the pAAV helper and pAAVrepcap8 plasmid, or the pAAV helper and pAAVrepcap9, or the pAAV helper and pAAV RH74 plasmid with calcium phosphate according to the manufacturer's instructions. Then, triple transfection was performed in HEK293T cells using the pAAV-shmiR plasmid (SR-construct) combined with one of the pAAV helper and capsid (pAAVrepcap8, pAAVrepcap9, or pAAVRH74). Next, HEK293T cells were cultured at 37°C in 5% CO2 for 72 hours. After lysing, the cells were purified by ultracentrifugation using an iodixanol (Sigma-Aldrich) step gradient, followed by ultracentrifugation using cesium chloride. The number of vector genomes was quantified by quantitative polymerase chain reaction (Q-PCR). 【0267】 [Example 3 - In vivo study using a single-vector "silence and substitution" approach] To test the in vivo efficacy of the SR-Construct described in Example 2 in a disease model associated with OPMD, the SR-Construct was individually administered to 10-12 week old A17 mice via intramuscular injection into the tibialis anterior (TA) muscle at doses within an arbitrary range. The dose was 7.5 × 10⁶ per muscle. 11 , 2.5×10 11 , 5×10 10 , 1 x 10 10 , 2×10 9 , and 4×10 8 The vector genome (vg) was used. Age-matched A17 mice injected with physiological saline were used as the untreated group. Mice were sacrificed at either 14 or 20 weeks post-treatment. 【0268】 [Example 4 - Quantitative measurement of shmiR production, PABPN1 silencing, and codon-optimized PABPN1 expression in A17 mice treated with SR-Construct] Fourteen weeks after SR-Construct treatment, TA muscle tissue was collected from A17 mice in Example 3, and RNA was extracted. SR-Construct-dependent expression of shmiR in TA muscle tissue was quantified (Figure 3A). The quantified expression levels of shmiR were dose-dependent of SR-Construct, as were the silencing of PABPN1 (expPABPN1, etc.) (Figure 3B) and the restoration of normal PABPN1 levels (Figure 3C). 【0269】 [Example 5 - Reduction in nuclear inclusion bodies (INIs) in A17 mice treated with SR-Construct] The effect of SR-Construct on the persistence of intranuclear inclusion bodies (INIs) was tested in A17 mice at 14 weeks of age in Example 3. FvB wild-type mice were also included as healthy control groups. Fourteen weeks after AAV injection, muscle tissue was collected and mounted for histological study. Sections were pre-treated with 1M KCl to preferentially elute all soluble PABPN1 from the tissue. Antimicrobial fluorescence detection of PABPN1 (green) and laminin (red), an abundant protein in the extracellular matrix of muscle cells, was detected in treated muscle sections, showing a significant reduction in the number of PABPN1-positive intranuclear inclusion bodies (INIs) in muscle treated with SR-Construct, with a dose-effect relationship (Figure 4A). Quantification of the proportion of nuclei containing INI in muscle sections shows that treatment with SR-Construct significantly reduces the amount of INI compared to untreated A17 muscle (one-way ANOVA with Bonferroni post-hoc test, ***p<0.001, ns: not significant) (Figure 4B). 【0270】 [Example 6 - Treatment with SR-Construct improves the physiological properties and function of the treated muscle.] The physiological properties and functionality of the treated muscles were measured in A17 mice at 14 weeks of age in Example 3. FvB wild-type mice were also included as healthy control groups. Maximum force generated by TA muscles was measured by in situ muscle physiology (Figure 5A). SR-Construct significantly increased the maximum force generated by TA muscles in a dose-dependent manner. Muscle weight normalized to body weight (BW) was also measured 14 weeks after SR-Construct administration (Figure 5B). Muscle weight normalized to body weight of SR-treated muscles was comparable to that of control FvB mice at doses exceeding approximately 1 e10 vg per injected TA (mean ± SEM n=10, one-way ANOVA with Bonferroni post-hoc test, *p<0.05, ***p<0.001, **p<0.01, ns: not significant). 【0271】 [Example 7 - Recovery of muscle function over time] Maximum force generated by the TA muscle of SR-Construct treated A17 mice and FvB wild-type mice was measured by in situ muscle physiology 14 weeks after SR-Construct administration (Figure 6A) and 20 weeks after SR-Construct administration (Figure 6B). At intermediate doses (1e10vg / TA and 6e10vg / TA), the beneficial effect on muscle strength was far more pronounced at 20 weeks compared to 14 weeks after injection (mean ± SEM n=10, one-way ANOVA with Bonferroni post-hoc test, ***p<0.001, **p<0.01). 【0272】 [Example 8 - Direct administration to the pharyngeal muscle of a sheep] Direct injection of SR-Construct into the pharyngeal muscle of sheep revealed that PABPN1, including all amino acid residues except for one at position 95, is highly conserved from sheep to humans. 【0273】 SR-Construct was injected directly into the pharyngeal muscle of sheep (Figure 7A). In the sheep study, two sheep were each injected with 1.5 e13 vg of SR-Construct into the cricopharyngeal muscle (CP), and then with an additional 1.0 e13 vg of SR-Construct into the pharyngeal muscle (pharynx). The remaining 10 animals treated with SR-Construct (1.0 e10 vg to 1.0 e13 vg) received injections only into the CP. A total of 1.5 ml was injected into the CP (three injections of 0.5 ml each). A total of 6 ml was injected into the pharynx (two injections of 1.5 ml each into both the right and left sides). 【0274】 Radioimaging using radiolabeled cream shows severe dysphagia in human OPMD patients at risk of aspiration (Figure 7B). 【0275】 [Example 9 - Design, Production, and Testing of Modified AAV VP1 Sequences] In this example, we designed and prepared AAVs containing specific sequence modifications, namely amino acid substitutions introduced into the phospholipase A2 (PLA2) domain and flanking sequences of the viral capsid protein subunit 1 (VP1), to restore AAV phospholipase activity and viral function when produced in insect cells. Furthermore, based on multiple alignments performed on VP1 subsequences containing the PLA2 domain and flanking sequences of various representative AAV serotypes, we prepared a consensus VP1 subsequence containing the PLA2 domain and flanking sequences, including sequence modifications designed to restore phospholipase activity. This wild-type AAV9 VP1 subsequence is described in Sequence ID No. 87. 【0276】 9.1 Design of the modified AAV9 VP1 sequence Sequence alignment was performed using the BLASTp alignment tool on the N-terminal 180 amino acids from the VP1 proteins of AAV9 (SEQ ID NO: 89), AAV8 (SEQ ID NO: 93), and AAV2 (SEQ ID NO: 97). Based on these alignments, it was shown that the PLA2 domain and adjacent sequences from AAV8 and AAV9 are highly conserved in the corresponding sequences within AAV2. Based on these sequence alignments, modified AAV9 VP1 and AAV8 VP1 sequences were designed in silico. The modified AAV9 VP1 sequences were designed by substituting the amino acids at positions 42, 67, 81, 84, and 85 of the sequence described in SEQ ID NO: 89 with the amino acids that occur at the corresponding positions in the AAV2 VP1 sequence described in SEQ ID NO: 97, namely A42S, A67E, Q81R, K84D, and A85S in the sequence of SEQ ID NO: 89. One of the substituted positions in the modified AAV9 VP1 sequence is located in a region adjacent to the PLA2 domain (but is likely involved in the folding and / or activity of the PLA2 domain), and four of the modified residue positions are located within the PLA2 domain itself. The modified AAV8 VP1 sequence was designed by substituting the amino acids at positions 42, 67, 81, 84, 85, and 105 of the sequence described in SEQ ID NO: 93 with the amino acids that would occur at the corresponding positions in the AAV2 VP1 sequence described in SEQ ID NO: 97, namely A42S, A67E, Q81R, K84D, A85S, and Q105K in the sequence of SEQ ID NO: 93. 【0277】 9.2 Production of baculoviral vectors expressing structural and unstructured AAV9 proteins Baculovirus vectors encoding AAV9 capsid proteins containing subunits VP1, VP2, and VP3, and AAV9 non-structural proteins Rep78, Rep68, Rep52, and Rep40 were prepared (BacAAV9-Rep-VPmod, Figure 8). Briefly, a DNA construct encoding AAV9 capsid proteins with a modified AAV9 VP1 subunit encoded by the sequence described in Sequence ID No. 90 and having adjacent NotI and ApaI restriction sites was synthesized using GenScript (AAV9-VPmod, Figure 9). The wtAAV9-Rep / Cap plasmid (Virovek, Hayward, CA), encoding non-structural proteins Rep78, Rep68, Rep52, and Rep40, as well as capsid proteins VP1, VP2, VP3, and assembly activation protein (AAP), was used as the backbone to accept the AAV9-VPmod DNA construct. Both the AAV9-VPmod DNA construct and the wtAAV9-Rep plasmid were digested with NotI and ApaI, respectively. Subsequently, the AAV9-VPmod DNA construct was ligated to the wtAAV9-Rep plasmid backbone in place of the wt capsid protein-coding sequence to generate the AAV9-Rep-VPmod (Figure 10). The AAV9-Rep-VPmod intermediate was then cloned into the pOET1 baculovirus transfer vector (Oxford Expression Technologies). To facilitate this, the EcoRV site was inserted into the AAV9-Rep-VPmod intermediate using the Quickchange technique to generate the AAV9-Rep-VPmod-EcoRV intermediate. Next, the AAV9-Rep-VPmod-EcoRV intermediate and pOET1 (Oxford Expression Technologies) were digested with NotI and EcoRV, respectively, and the insert was ligated to the pOET1 skeleton to generate the final AAV9-Rep-VPmod clone (BacAAV9-Rep-CapPL, Figure 8). 【0278】 9.3 Production of baculoviral vectors expressing structural and unstructured AAV8 proteins Baculovirus vectors encoding modified AAV8 capsid proteins containing subunits VP1, VP2, and VP3, and AAV8 non-structural proteins Rep78 and Rep52 were prepared (BacAAV8-Rep-VPmod, Figure 11). Briefly, a DNA construct encoding AAV8 capsid proteins (VP1, VP2, and Vp3) containing the sequence described in Sequence ID No. 94 and a modified VP1 subunit with adjacent NotI and ApaI restriction sites was synthesized using GenScript (AAV8-VPmod, Figure 12). The wtAAV8-Rep / Cap plasmid (Virovek, Hayward, CA), encoding non-structural proteins Rep78, Rep68, Rep52, and Rep40, as well as capsid proteins VP1, VP2, VP3, and assembly activation protein (AAP), was used as the backbone to accept the AAV8-VPmod DNA construct. Both the AAV8-VPmod DNA construct and the wtAAV8-Rep / Cap plasmid were digested with NotI and ApaI, respectively. Subsequently, the AAV8-VPmod DNA construct was ligated to the wtAAV8-Rep / Cap plasmid backbone in place of the wt capsid protein-coding sequence to generate the AAV8-Rep-VPmod (Figure 13). The AAV8-Rep-VPmod intermediate was then cloned into the pOET1 baculovirus transfer vector (Oxford Expression Technologies). To facilitate this, the EcoRV site was inserted into the AAV8-Rep-VPmod intermediate using the Quickchange technique to generate the AAV8-Rep-VPmod-EcoRV intermediate. Next, the AAV8-Rep-VPmod-EcoRV intermediate and pOET1 were digested with NotI and EcoRV, and the insert was ligated to the pOET1 skeleton (Oxford Expression Technologies) to produce the final AAV8-Rep-VPmod clone (BacAAV8-Rep-VPmod, Figure 11). 【0279】 9.4 Production of a baculovirus vector expressing the target gene (GOI) Baculovirus vectors encoding target genes (GOIs) adjacent to the AAV2 reverse terminal repeat (ITR) were prepared. Briefly, in one example, a DNA construct encoding two shmiRs targeting the human PABPN1 transcript adjacent to the AAV2 ITR was cloned into the pOET1 baculovirus transfer vector (Oxford Expression Technologies) by digesting the AAV2-GOI construct (Figure 14) and pOET1 (Oxford Expression Technologies) with NotI, ligating the AAV2-GOI construct to the pOET1 backbone, and generating the final clone (BacAAV2-GOI, Figure 15). The second GOI, encoding three shmiRs targeting various regions of the HBV polymerase gene transcript, was also prepared in the same manner as described above. 【0280】 9.5 Generation of P0 baculovirus strains The baculovirus P0 strain was generated using the baculoCOMPLETE system (Oxford Expression Technologies) (according to manufacturer's instructions). Briefly, 1 million Sf9 cells were seeded into a 6-well plate and adhered to the plate 1 hour before transfection. 500 ng of Bac-AAV2-GOI plasmid, BacAAV8-Rep-CapPL, or BacAAV9-Rep-CapPL was mixed with 500 ng of flush BAC DNA and baculoFECTIN transfection reagent in 1 ml of TC100 medium (according to manufacturer's protocol). After incubation at room temperature for 30 minutes, the transfection mixture was added to the seeded Sf9 cells. The 6-well plate was incubated at 28°C. 24 hours after transfection, 1 ml of Sf9 medium was added to the cells. Five days after transfection, the medium containing the P0 baculovirus strain was collected and stored at 4°C. Therefore, P0 baculovirus was produced in response to BacAAV8-Rep-CapPL, BacAAV9-Rep-CapPL, and Bac-AAV2-GOI. 【0281】 9.6 AAV prepared in mammalian cells The functionality of AAV prepared in mammalian cells was compared with that of AAV prepared in insect cells as described above. To compare the biological activity (functionality) of recombinant AAV prepared in mammalian and insect cells, mammalian cells were infected with viruses of various titers in vitro, and the expression of shmiR after processing was quantified using a qRTPCR assay. 【0282】 For these experiments, recombinant AAV8 particles expressing three shmiRs targeting the HBV polymerase gene transcript were prepared in mammalian cells by a commercial supplier (Vector Biolabs; https: / / www.vectorbiolabs.com). Furthermore, recombinant AAV9 particles expressing two shmiRs targeting human PABPN1 were prepared by a second supplier of mammalian cells, Nationwide Children's Hospital Vector Core (https: / / www.nationwidechildrens.org / research / resources-infrastructure / core-facilities / viralvector-core-clinical-manufacturing-facility). 【0283】 The biological activity was evaluated for (i) AAV8 containing unmodified VP1 produced in mammalian cells (Vector Biolabs), (ii) AAV8 containing modified VP1 produced by baculovirus in insect cells (using BacAAV8-Rep-VPmod as described herein), and (iii) AAV8 containing unmodified wtVP1 produced by baculovirus in insect cells using wtAAV8-Rep / Cap (Ben10, Virovek, Hayward, CA). Each of these encodes three shmiRs that target the HBV polymerase gene (HBV shmiRs are referred to as all-4_m3, shRNA8v2_p1, and All-9_p1). In short, JHU67 cells were infected with the modified or unmodified recombinant virus preparations described above at MOIs of 4x10e9, 8x10e9, and 1.6x10e10, and shmiR expression was quantified for each of the three shmiRs 72 hours after infection. To quantify shmiR expression, RNA was extracted from infected cells using the Qiagen RNA mini-kit (Qiagen). The RNA was reverse transcribed using the Qiagen miScript kit (Qiagen). The cDNA was then used in a qPCR reaction with specific primers designed to amplify the shmiR target to determine the total number of copies present in the sample. 【0284】 As shown in Figures 16A to 16C, cells infected with AAV8 containing unmodified wt VP1 prepared in mammalian cells produced easily detectable levels of shmiR, while AAV8 containing unmodified wt VP1 produced by baculovirus in insect cells produced almost no shmiR. In contrast, AAV8 containing modified VP1 produced by baculovirus in insect cells produced relatively high levels of shmiR, indicating enhanced function of these AAVs compared to AAV8 containing unmodified wtVP1 produced by baculovirus in insect cells. 【0285】 The biological activity was also evaluated for (i) AAV9 with an unmodified capsid protein produced in mammalian cells (nationwide), and (ii) AAV9 with a modified capsid protein produced by baculovirus using BACAAV9-Rep-VPmod (as described herein) in insect cells, each encoding two shmiRs (referred to as sh13 and sh17) that target the human PABPN1 transcript. Briefly, C2C12 cells expressing the AAV endogenized receptor were infected with 4x10e9, 8x10e9, and 1.6x10e10 vector genomes. After 72 hours of incubation, the cells were harvested, RNA was extracted, and the shmiR expression of the two shmiRs was quantified according to the qPCR method described above. 【0286】 As shown in Figure 17, the two preparations exhibited very similar levels of shmiR expression, which indicates very similar viral function. 【0287】 Although demonstrated in the context of AAVs from serotypes 8 and 9, it is believed that AAV function can be restored when produced from a baculovirus expression system in insect cells by modifying the VP1 subunit sequence of other AAV serotypes (other than serotype 2) according to the approach described herein. 【0288】 Those skilled in the art will understand that numerous variations and / or modifications can be made to the above embodiments without departing from the broad general scope of this disclosure. Accordingly, these embodiments should be considered in all respects illustrative and not limiting. This application provides the following: 1. (a) AAV9-derived viral capsid protein containing a modified subunit 1 (VP1) sequence in which the amino acids at positions 1, 26, 40, 43, and 44 are modified compared to the corresponding wild-type AAV9 VP1 sequence described in Sequence ID No. 87: and (b) an adeno-associated virus (AAV) comprising a polynucleotide sequence comprising (i) a ddRNAi construct comprising a nucleic acid comprising a sequence encoding a short hairpin microRNA (shmiR); and (ii) a PABPN1 construct comprising a nucleic acid comprising a sequence encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR(s) encoded by the ddRNAi construct. 2. The AAV described in 1 above, wherein the modified VP1 sequence contains serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and serine at position 44, compared to the wild-type AAV9 VP1 sequence described in Sequence ID No. 87. 3. The AAV according to 1 or 2 above, wherein the modified AAV9 VP1 sequence includes the sequence described in sequence number 88. 4. The AAV according to any one of 1 to 3 above, wherein the polynucleotide sequence includes the ddRNAi construct and the PABPN1 construct in the 5' to 3' direction. 5. The AAV according to any one of 1 to 3 above, wherein the polynucleotide sequence includes the PABPN1 construct and the ddRNAi construct in the 5' to 3' direction. 6. The AAV according to any one of 1 to 5 above, wherein the polynucleotide sequence of (b) includes a reverse terminal repeat sequence (ITR) from the AAV serotype, and the ITR is adjacent to the sequence containing the ddRNAi construct and the PABPN1 construct. 7. The AAV described in 6 above, wherein the ITR is derived from the AAV2 serotype. 8. The AAV according to any one of 1 to 7 above, wherein the sequence encoding the functional PABPN1 protein is codon-optimized so that its mRNA transcript is not targeted by the shmiR of the ddRNAi construct. 9. The sequence encoding the functional PABPN1 protein is the AAV described in SEQ ID NO: 73, or any of the AAVs described in 1 to 8 above. 10. The AAV according to any one of 1 to 9 above, wherein the ddRNAi construct and the sequence encoding the functional PABPN1 protein are operably linked to a promoter located upstream of the ddRNAi construct and the sequence encoding the functional PABPN1 protein. 11. The AAV described in 10 above, wherein the promoter is a muscle-specific promoter. 12. The aforementioned shmiR, An effector sequence with a length of at least 17 nucleotides; Complementary arrangement of effectors; Stem-loop array; and Includes a primary microRNA (pri-miRNA) backbone; The AAV according to any one of 1 to 11, wherein the effector sequence is substantially complementary to a region of the corresponding length in the RNA transcript described in any one of sequence numbers 1 to 13. 13. The aforementioned shmiR, shmiR; including the effector sequence described in Sequence ID No. 15 and the effector complementary sequence described in Sequence ID No. 14. shmiR; including the effector sequence described in Sequence ID No. 17 and the effector complementary sequence described in Sequence ID No. 16; shmiR; including the effector sequence described in SEQ ID NO: 19 and the effector complementary sequence described in SEQ ID NO: 18. shmiR; including the effector sequence described in Sequence ID No. 21 and the effector complementary sequence described in Sequence ID No. 20. shmiR; including the effector sequence described in Sequence ID 23 and the effector complementary sequence described in Sequence ID 22; shmiR; including the effector sequence described in Sequence ID No. 25 and the effector complementary sequence described in Sequence ID No. 24. shmiR; including the effector sequence described in Sequence ID No. 27 and the effector complementary sequence described in Sequence ID No. 26. shmiR; including the effector sequence described in Sequence ID No. 29 and the effector complementary sequence described in Sequence ID No. 28. shmiR; including the effector sequence described in Sequence ID 31 and the effector complementary sequence described in Sequence ID 30; shmiR; including the effector sequence described in Sequence ID 33 and the effector complementary sequence described in Sequence ID 32. shmiR; including the effector sequence described in Sequence ID 35 and the effector complementary sequence described in Sequence ID 34. shmiR; including the effector sequence described in Sequence ID 37 and the effector complement sequence described in Sequence ID 36, and An AAV according to any of 1 to 12 above, selected from the group consisting of shmiR including the effector sequence described in Sequence ID No. 39 and the effector complementary sequence described in Sequence ID No. 38. 14. The shmiR is in the direction from 5' to 3', The 5' adjacent sequence of the pri-miRNA skeleton; The aforementioned complementary array of effectors; The aforementioned stem-loop array; The aforementioned effector arrangement; and An AAV according to any one of the above 1 to 13, comprising the 3' adjacent sequence of the pri-miRNA skeleton. 15. The AAV described in 14 above, wherein the stem-loop sequence is the sequence described in sequence number 40. 16. The AAV described in 14 or 15 above, wherein the pri-miRNA skeleton is a pri-miR-30a skeleton. 17. An AAV according to any one of 14 to 16 above, wherein the 5' adjacent sequence of the pri-miRNA skeleton is described in SEQ ID NO: 41, and the 3' adjacent sequence of the pri-miRNA skeleton is described in SEQ ID NO: 42. 18. The AAV according to any one of 1 to 17 above, wherein the ddRNAi construct comprises at least two nucleic acids, each encoding a shmiR, and each shmiR comprises an effector sequence substantially complementary to the RNA transcript corresponding to the PABPN1 protein that causes OPMD, and each shmiR comprises a different effector sequence. 19. An AAV according to any one of 1 to 18 above, wherein each of the at least two nucleic acids encodes a shmiR comprising an effector sequence substantially complementary to a region of corresponding length in the RNA transcript described in one of Sequence IDs 1, 2, 4, 7, 9, 10, and 13. 20. The at least two nucleic acids are A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR2) including the effector sequence described in SEQ ID NO: 15 and the effector complement sequence described in SEQ ID NO: 14; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR3) including the effector sequence described in SEQ ID NO: 17 and the effector complement sequence described in SEQ ID NO: 16; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR5) including the effector sequence described in SEQ ID NO: 21 and the effector complement sequence described in SEQ ID NO: 20; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR9) including the effector sequence described in SEQ ID NO: 27 and the effector complement sequence described in SEQ ID NO: 26; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR13) including the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30; nucleic acids comprising or consisting of DNA sequences encoding shmiR(shmiR14) including the effector sequence described in SEQ ID NO: 33 and the effector complement sequence described in SEQ ID NO: 32; and The AAV according to 19 above, selected from the group consisting of nucleic acids comprising a DNA sequence encoding shmiR(shmiR17) including the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO: 38. 21. The at least two nucleic acids are Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 56 (shmiR2); Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 57 (shmiR3); Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 59 (shmiR5); Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 62 (shmiR9); Nucleic acids containing or consisting of the DNA sequence described in SEQ ID NO: 64 (shmiR13); Nucleic acids containing or consisting of the DNA sequence described in SEQ ID NO: 65 (shmiR14); and The AAV described in item 19 above, selected from the group consisting of nucleic acids containing or comprising the DNA sequence described in Sequence ID No. 68 (shmiR17). 22. The AAV according to 19, wherein each of the at least two nucleic acids encodes a shmiR comprising an effector sequence substantially complementary to a region of corresponding length in the RNA transcript described in one of Sequence IDs 2, 9, 10, and 13. 23. The at least two nucleic acids are A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR3) including the effector sequence described in SEQ ID NO: 17 and the effector complement sequence described in SEQ ID NO: 16; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR13) including the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30; nucleic acids comprising or consisting of DNA sequences encoding shmiR(shmiR14) including the effector sequence described in SEQ ID NO: 33 and the effector complement sequence described in SEQ ID NO: 32; and The AAV according to 19 above, selected from the group consisting of nucleic acids comprising a DNA sequence encoding shmiR(shmiR17) including the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO: 38. 24. The at least two nucleic acids are Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 57 (shmiR3); Nucleic acids containing or consisting of the DNA sequence described in SEQ ID NO: 64 (shmiR13); Nucleic acids containing or consisting of the DNA sequence described in SEQ ID NO: 65 (shmiR14); and The AAV described in item 19 above, selected from the group consisting of nucleic acids containing or comprising the DNA sequence described in Sequence ID No. 68 (shmiR17). 25. The ddRNAi construct is (a) nucleic acids comprising or consisting of a DNA sequence encoding shmiR, which includes the effector sequence described in SEQ ID NO: 31 and the effector complement sequence (shmiR13) described in SEQ ID NO: 30; and (b) AAV according to any one of 1 to 24 above, comprising a DNA sequence encoding shmiR including the effector sequence described in SEQ ID NO: 39 and the effector complement sequence (shmiR17) described in SEQ ID NO: 38, or a nucleic acid consisting thereof. 26. The ddRNAi construct is (a) nucleic acids comprising or consisting of the DNA sequence described in Sequence ID No. 64 (shmiR13); (b) The AAV according to 25, comprising a nucleic acid comprising or consisting of the DNA sequence described in Sequence ID No. 68 (shmiR17). 27. A pharmaceutical composition comprising an AAV as described in any of items 1 to 26 above, and one or more pharmaceutically acceptable carriers. 28. A method for treating a subject suffering from oculopharyngeal muscular dystrophy (OPMD), comprising administering to the subject an AAV described in any of items 1 to 26 above or a pharmaceutical composition described in item 27 above. 29. The method according to 28, wherein the composition is administered by direct injection into the pharyngeal muscle of the subject. 30. The method according to 29, wherein the pharyngeal muscles include one or more of the inferior pharyngeal muscles, middle pharyngeal muscles, superior pharyngeal constrictor muscles, palatinaryngeal muscles, tubal pharyngeal muscles, stylopharyngeal muscles, or any combination thereof.

Claims

[Claim 1] (a) AAV9-derived viral capsid protein comprising a modified subunit 1 (VP1) sequence containing any five or more of the following as defined for the corresponding wild-type AAV9 VP1 subsequence described in Sequence ID No. 87: serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, serine at position 44, and / or lysine at position 64, wherein only any four or more of the amino acids at positions 1, 26, 40, 43, and 44 are substituted for the corresponding wild-type AAV9 VP1 sequence: and (b) (i) a DNA-directed RNAi (ddRNAi) construct comprising a polynucleotide encoding a short hairpin microRNA (shmiR); and (ii) a PABPN1 construct comprising a polynucleotide encoding a functional PABPN1 protein translated from an mRNA transcript not targeted by the shmiR(s) encoded by the ddRNAi construct, comprising a polynucleotide Adeno-associated viruses (AAVs), including adeno-associated viruses. [Claim 2] (a) AAV9-derived viral capsid protein comprising a modified subunit 1 (VP1) sequence containing glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, serine at position 44, and / or lysine at position 64 as defined for the corresponding wild-type AAV9 VP1 subsequence described in Sequence ID No. 87, wherein the amino acids at positions 26, 40, 43, and 44 are substituted for those in the corresponding wild-type AAV9 VP1 sequence: and (b) (i) a DNA-directed RNAi (ddRNAi) construct comprising a polynucleotide encoding a short hairpin microRNA (shmiR); and (ii) a PABPN1 construct comprising a polynucleotide encoding a functional PABPN1 protein translated from an mRNA transcript not targeted by the shmiR(s) encoded by the ddRNAi construct, comprising a polynucleotide The AAV according to claim 1, including the AAV described in claim 1. [Claim 3] The AAV according to claim 1 or 2, wherein the modified VP1 sequence includes the amino acid sequence described in SEQ ID NO: 87, except that the amino acid substitutions A26E, Q40R, K43D, and A44S are removed from the amino acid sequence described in SEQ ID NO:

87. [Claim 4] The AAV according to claim 1 or 2, wherein the modified VP1 sequence contains glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and serine at position 44, as defined for the corresponding wild-type AAV9 VP1 sequence described in Sequence ID No.

87. [Claim 5] The AAV according to claim 1 or 2, wherein the modified VP1 sequence comprises serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and serine at position 44, as defined for the corresponding wild-type AAV9 VP1 sequence described in Sequence ID No.

87. [Claim 6] The AAV according to claim 1, wherein the modified AAV9 VP1 sequence includes the sequence described in sequence number 88. [Claim 7] (i)(b) The polynucleotide contains the ddRNAi construct and the PABPN1 construct in the 5' to 3' direction, or The polynucleotide of (ii)(b) comprises the PABPN1 construct and the ddRNAi construct in the 5' to 3' direction, The AAV according to any one of claims 1 to 6. [Claim 8] The AAV according to any one of claims 1 to 7, wherein the polynucleotide in (b) comprises a reverse terminal repeat sequence (ITR) from the AAV serotype, and the ITR is adjacent to the polynucleotide comprising the ddRNAi construct and the PABPN1 construct. [Claim 9] The AAV according to claim 8, wherein the ITR is derived from the AAV2 serotype. [Claim 10] (i) The polynucleotide encoding the functional PABPN1 protein is codon-optimized such that its mRNA transcript is not targeted by the shmiR of the ddRNAi construct, (ii) The polynucleotide encoding the functional PABPN1 protein is described in Sequence ID No. 73, (iii) The ddRNAi construct and the polynucleotide encoding the functional PABPN1 protein are operably linked to a promoter located upstream of the ddRNAi construct and the polynucleotide encoding the functional PABPN1 protein, or (iv) Any combination of (i) through (iii), The AAV according to any one of claims 1 to 9. [Claim 11] The AAV according to claim 10, wherein the promoter is a muscle-specific promoter. [Claim 12] The aforementioned shmiR, An effector sequence with a length of at least 17 nucleotides; Complementary arrangement of effectors; Stem-loop array; and It contains a primary microRNA (prim-miRNA) backbone; The AAV according to any one of claims 1 to 11, wherein the effector sequence is complementary to a region of the corresponding length in the RNA transcript described in any one of SEQ ID NOs: 13 and 1 to 12, or complementary to a region of the corresponding length in the RNA transcript described in any one of SEQ ID NOs: 13 and 1 to 12, with the exception of 4, 3, 2, or 1 mismatch with any one of SEQ ID NOs: 13 and 1 to 12. [Claim 13] The aforementioned shmiR, shmiR including the effector sequence described in Sequence ID 39 and the effector complementary sequence described in Sequence ID 38; shmiR including the effector sequence described in Sequence ID No. 15 and the effector complementary sequence described in Sequence ID No. 14; shmiR including the effector sequence described in Sequence ID No. 17 and the effector complementary sequence described in Sequence ID No. 16; shmiR including the effector sequence described in Sequence ID No. 19 and the effector complementary sequence described in Sequence ID No. 18; shmiR including the effector sequence described in SEQ ID NO: 21 and the effector complement sequence described in SEQ ID NO: 20; shmiR including the effector sequence described in Sequence ID No. 23 and the effector complement sequence described in Sequence ID No. 22; shmiR including the effector sequence described in Sequence ID No. 25 and the effector complement sequence described in Sequence ID No. 24; shmiR including the effector sequence described in Sequence ID No. 27 and the effector complementary sequence described in Sequence ID No. 26; shmiR including the effector sequence described in Sequence ID No. 29 and the effector complementary sequence described in Sequence ID No. 28; shmiR including the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30; shmiR including the effector sequence described in SEQ ID NO: 33 and the effector complement sequence described in SEQ ID NO: 32; shmiR including the effector sequence described in Sequence ID No. 35 and the effector complement sequence described in Sequence ID No. 34; and shmiR including the effector sequence described in Sequence ID 37 and the effector complement sequence described in Sequence ID 36 An AAV according to any one of claims 1 to 12, selected from the group consisting of the following. [Claim 14] The AAV according to any one of claims 1 to 13, wherein each ddRNAi construct comprises at least two nucleic acids encoding shmiR, each shmiR comprising an effector sequence complementary to an RNA transcript corresponding to the PABPN1 protein causing OPMD, or an effector sequence complementary to an RNA transcript corresponding to the PABPN1 protein causing OPMD, with 4, 3, 2, or 1 mismatch removed from the RNA transcript corresponding to the PABPN1 protein causing OPMD, and each shmiR comprises a different effector sequence. [Claim 15] Each shmiR is directed from 5' to 3', (i) 5' adjacent sequence of the prim-miRNA backbone; Complementary arrangement of effectors; Stem loop array; Effector arrangement; and It contains the 3' adjacent sequence of the prim-miRNA backbone, or (ii) 5' adjacent sequence of the prim-miRNA backbone; Effector arrangement; Stem loop array; Effector complementary array; and Includes the 3' adjacent sequence of the prim-miRNA backbone, The AAV according to any one of claims 1 to 14. [Claim 16] (i) Whether the stem-loop sequence is the sequence described in sequence number 40, (ii) Whether the pri-miRNA skeleton is a pri-miR-30a skeleton, (iii) The 5' adjacent sequence of the prim-miRNA skeleton is described in SEQ ID NO: 41, and the 3' adjacent sequence of the prim-miRNA skeleton is described in SEQ ID NO: 42, or (iv) Any combination of (i) through (iii), The AAV according to claim 15. [Claim 17] The ddRNAi construct comprises at least two nucleic acids, each encoding a shmiR, and each shmiR is An effector sequence with a length of at least 17 nucleotides; Complementary arrangement of effectors; Stem-loop array; and It contains a primary microRNA (prim-miRNA) backbone; The AAV according to any one of claims 1 to 16, wherein each shmiR includes an effector sequence that is complementary to a region of the corresponding length in the RNA transcript described in one of sequence numbers 1, 2, 4, 7, 9, 10, and 13, or that is complementary to a region of the corresponding length in the RNA transcript described in one of sequence numbers 1, 2, 4, 7, 9, 10, and 13, with 4, 3, 2, or 1 mismatch to one of sequence numbers 1, 2, 4, 7, 9, 10, and 13. [Claim 18] (i) The at least two nucleic acids A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR2) including the effector sequence described in SEQ ID NO: 15 and the effector complement sequence described in SEQ ID NO: 14; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR3) including the effector sequence described in SEQ ID NO: 17 and the effector complement sequence described in SEQ ID NO: 16; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR5) including the effector sequence described in SEQ ID NO: 21 and the effector complement sequence described in SEQ ID NO: 20; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR9) including the effector sequence described in SEQ ID NO: 27 and the effector complement sequence described in SEQ ID NO: 26; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR13) including the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30; nucleic acids comprising or consisting of DNA sequences encoding shmiR (shmiR14) including the effector sequence described in SEQ ID NO: 33 and the effector complement sequence described in SEQ ID NO: 32; and A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR17) which includes the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO:

38. Selected from the group consisting of, (ii) The at least two nucleic acids, Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 56 (shmiR2); Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 57 (shmiR3); Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 59 (shmiR5); Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 62 (shmiR9); Nucleic acids containing or consisting of the DNA sequence described in SEQ ID NO: 64 (shmiR13); Nucleic acids containing or consisting of the DNA sequence described in SEQ ID NO: 65 (shmiR14); and Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 68 (shmiR17) Selected from the group consisting of, The AAV according to claim 17. [Claim 19] (i) Each of the at least two nucleic acids is complementary to a region of the corresponding length in the RNA transcript described in one of SEQ ID NOs: 2, 9, 10, and 13, or encodes a shmiR that includes an effector sequence complementary to a region of the corresponding length in the RNA transcript described in one of SEQ ID NOs: 2, 9, 10, and 13, with 4, 3, 2, or 1 mismatch to one of SEQ ID NOs: 2, 9, 10, and 13. (ii) The at least two nucleic acids, A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR3) including the effector sequence described in SEQ ID NO: 17 and the effector complement sequence described in SEQ ID NO: 16; A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR13) including the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30; nucleic acids comprising or consisting of DNA sequences encoding shmiR (shmiR14) including the effector sequence described in SEQ ID NO: 33 and the effector complement sequence described in SEQ ID NO: 32; and A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR17) which includes the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO:

38. Selected from the group consisting of, (iii) The at least two nucleic acids, Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 57 (shmiR3); Nucleic acids containing or consisting of the DNA sequence described in SEQ ID NO: 64 (shmiR13); Nucleic acids containing or consisting of the DNA sequence described in SEQ ID NO: 65 (shmiR14); and Nucleic acids containing or consisting of the DNA sequence described in Sequence ID No. 68 (shmiR17) Selected from the group consisting of, The AAV according to claim 17. [Claim 20] The aforementioned ddRNAi construct (a) a nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR13) which includes the effector sequence described in SEQ ID NO: 31 and the effector complement sequence described in SEQ ID NO: 30; and (b) A nucleic acid comprising or consisting of a DNA sequence encoding shmiR (shmiR17) which includes the effector sequence described in SEQ ID NO: 39 and the effector complement sequence described in SEQ ID NO:

38. AAV according to any one of claims 1 to 19, including the AAV described in any one of claims 1 to 19. [Claim 21] The aforementioned ddRNAi construct, (a) nucleic acids comprising or consisting of the DNA sequence described in Sequence ID No. 64 (shmiR13); and (b) A nucleic acid comprising or consisting of the DNA sequence described in Sequence ID No. 68 (shmiR17) The AAV according to claim 20, including the AAV described in claim 20. [Claim 22] A pharmaceutical composition comprising an AAV according to any one of claims 1 to 21 and one or more pharmaceutically acceptable carriers. [Claim 23] An AAV according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 22, for treating a subject suffering from oculopharyngeal muscular dystrophy (OPMD), or for preventing OPMD in a subject with a predisposition to OPMD. [Claim 24] Use of an AAV according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 22 in the preparation of a pharmaceutical for treating a subject suffering from oculopharyngeal muscular dystrophy (OPMD) or for preventing OPMD in a subject with a predisposition to OPMD.

Images